Adeno-associated virus vector variants for high efficiency genome editing and methods thereof

ABSTRACT

Adeno-associated virus (AAV) Clade F vectors or AAV vector variants (relative to AAV9) for precise editing of the genome of a cell and methods and kits thereof are provided. Targeted genome editing using the AAV Clade F vectors or AAV vector variants provided herein occurred at frequencies that were shown to be 1,000 to 100,000 fold more efficient than has previously been reported. Also provided are methods of treating a disease or disorder in a subject by editing the genome of a cell of the subject via transducing the cell with an AAV Clade F vector or AAV vector variant as described herein and further transplanting the transduced cell into the subject to treat the disease or disorder of the subject. Also provided herein are methods of treating a disease or disorder in a subject by in vivo genome editing by directly administering the AAV Clade F vector or AAV vector variant as described herein to the subject.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/858,199, filed Apr. 24, 2020, which is a continuation of U.S. patentapplication Ser. No. 16/543,072, filed Aug. 16, 2019, which is acontinuation of U.S. patent application Ser. No. 15/894,538, filed Feb.12, 2018, now U.S. Pat. No. 10,443,075, which is a divisional of U.S.patent application Ser. No. 15/273,892, filed Sep. 23, 2016, now U.S.Pat. No. 9,890,396, which is a continuation of International PatentApplication No. PCT/US2015/051785, filed Sep. 23, 2015, which claims thebenefit of the priority date of U.S. Provisional Patent Application Ser.No. 62/209,862, filed Aug. 25, 2015, of U.S. Provisional PatentApplication Ser. No. 62/063,587, filed Oct. 14, 2014, and of U.S.Provisional Patent Application Ser. No. 62/054,899, filed Sep. 24, 2014.The contents of each of these referenced applications are incorporatedherein by reference in their entirety.

STATEMENT OF GOVERNMENT INTEREST

The present invention was made with government support under Grant No.HL087285 awarded by the National Institutes of Health. The Governmenthas certain rights in the invention.

SEQUENCE LISTING

The content of the electronically submitted sequence listing in ST.26format (Name: “404217-HMW-005D1C3_192782.xml”; Size: 112,977 bytes; andDate of Creation: Jul. 26, 2022) is incorporated herein by reference inits entirety.

BACKGROUND

The adeno-associated virus (AAV) genome is built of single-strandeddeoxyribonucleic acid (ssDNA), either positive- or negative-sensed,which is about 4.9 kilobase long. The genome comprises inverted terminalrepeats (ITRs) at both ends of the DNA strand, and two open readingframes (ORFs): rep and cap. Rep is composed of four overlapping genesencoding rep proteins required for the AAV life cycle, and cap containsoverlapping nucleotide sequences of capsid proteins: VP1, VP2 and VP3,which interact together to form a capsid of an icosahedral symmetry.

Recombinant adeno-associated virus (rAAV) vectors derived from thereplication defective human parvovirus AAV2 are proving to be safe andeffective gene transfer vehicles that have yet to be definitivelyidentified as either pathogenic or oncogenic [3-4, 6, 18-19, 26, 31].rAAV transduce non-dividing primary cells, are low in immunogenicity,and direct sustained transgene expression in vivo [6, 10, 20]. Infectionwith wild type AAV is associated with inhibition of oncogenictransformation and AAV inverted terminal repeats may actually conferoncoprotection [2, 28, 52-55]. A recent survey of panels of humantissues found that the marrow and liver were the two most common sitesof naturally occurring AAV isolates in humans, suggesting that infectionof marrow cells by AAV is not rare.

Use of viral vectors for gene therapy has been long considered. Due toits potential for long-lived correction and the ease of ex vivomanipulation, the hematopoietic system was one of the earliest targetsof gene therapy. Despite significant effort, however, actual therapeuticsuccess remains elusive [5]. This is due to the recognized inability ofmost viral vectors to efficiently transduce quiescent, non-dividinghematopoietic stem cells (HSC) [23] as well as safety concerns arisingfrom insertional oncogenesis [15, 22]. However, stable gene transfer hasbeen successfully demonstrated to both murine and human HSC by rAAV [8,11-12, 24, 27, 29-30, 37].

It has been additionally difficult to effectively use viral vectors ingene therapy for treating neurological conditions, particularly centralnervous system diseases or disorders due to the difficulty of crossingthe blood-brain barrier, a cellular and metabolic separation of thecirculating blood from the brain extracellular fluid created by tightjunctions between endothelial cells that restrict the passage ofsolutes.

CD34 is cell surface glycoprotein and a cell-cell adhesion factor. CD34protein is expressed in early hematopoietic and vascular tissue and acell expressing CD34 is designated CD34⁺. Chromosomal integration ofrAAV in human CD34⁺ HSC [8, 12, 16, 29] and efficient transduction ofprimitive, pluripotent, self-renewing human HSC capable of supportingprimary and secondary multi-lineage engraftment has been demonstrated inimmune-deficient NOD-SCID mice [29]. Transduction of primitive HSCcapable of supporting serial engraftment was shown to be attributable tothe propensity of rAAV to efficiently transduce primitive, quiescentCD34+CD38− cells residing in GO [24]. Despite several reports ofsuccessful rAAV-mediated gene transfer into human HSC in vitro and inmurine and non-human primate HSC in vivo, controversy regarding theutility of rAAV for HSC transduction still persists. These discrepanciesarose primarily from short-term in vitro studies that assessedtransduction by expression profiling and are attributable to theidentified restrictions to transgene expression from rAAV2, includingviral uncoating [35], intracellular trafficking [33], nuclear transportand second strand synthesis [36].

While AAV2 remains the best-studied prototypic virus for AAV-basedvectors [1, 13, 18, 21], the identification of a large number of new AAVserotypes significantly enhances the repertoire of potential genetransfer vectors [14]. AAV1, 3 and 4 were isolated as contaminants ofadenovirus stocks, and AAV5 was isolated from a human condylomatouswart. AAV6 arose as a laboratory recombinant between AAV1 and AAV2.Recently, more than 100 distinct isolates of naturally occurring AAV inhuman and non-human primate tissues were identified. This led to the useof capsids derived from some of these isolates for pseudotyping,replacing the envelope proteins of AAV2 with the novel envelopes,whereby rAAV2 genomes are then packaged using AAV2 rep and novel capsidgenes. The use of novel capsids, the proteins as part of the viralshell, resulted in the circumvention of many limitations in transgeneexpression associated with AAV2 [32, 35-36].

In an effort to circumvent these restrictions, recent research has shownthat novel capsid sequences result in reduced proteasome-mediated capsiddegradation, increased nuclear trafficking and retention. Novel capsids,many of which utilize novel receptors, broadens the tropism of rAAVallowing for efficient transduction of previously refractory tissues andprovides a means of circumventing highly prevalent pre-existingserologic immunity to AAV2, which posed major clinical limitations in arecent trial. Notably, some novel capsids appear to alter theintracellular processing of rAAV. For example, uncoating and transgeneexpression is accelerated in the context of AAV8 as compared to nativeAAV2 capsids. Recently, transgene expression was shown to be based uponcapsid proteins, regardless of the serotype origin of the invertedterminal repeats (ITRs).

Naturally occurring AAV is identifiable in cytokine-primed peripheralblood stem cells. Capsid sequences of these AAV are unique. Thesecapsids are capable of pseudotyping recombinant AAV2 genomes. US PatentPublication Number 20130096182A1 describes capsids AAVF1-17, and usethereof for cell transduction and gene transfer. Any improvement in thearea of gene therapy regarding both permanent and reversible genetransfer and expression for therapeutic purposes would be a significantimprovement in the art. Moreover, safe and efficient gene delivery tostem cells remains a significant challenge in the field despite decadesof research. Therefore the ability to genetically modify stem cellssafely would represent a significant advance.

Further, genome editing by gene targeting or correction at a specificsite in the genome without leaving a footprint in the genome isattractive for the precise correction of inherited and acquireddiseases. Current technology accomplishes this through the use ofexogenous endonucleases such as zinc finger nucleases, TAL endonucleasesor caspase 9/CRISPR systems. However, these “traditional” approaches areassociated with toxicity and off target effects of endonucleasecleavage. Therefore, the ability to genetically modify stem cells safelyand efficiently at high frequencies without the need for exogenousendonuclease cleavage would represent a significant advance.

Additionally, current methods of genetic transduction of human HSCsinvolve ex vivo transduction of purified donor stem cells followed bytransplantation into usually “conditioned” recipients. The cell harvestprocedures are invasive and involve either bone marrow harvest ormultiple days of granulocyte-colony stimulating factor (G-CSF) primingof the donor followed by apheresis. The ex vivo transduction procedurescan affect the hematopoietic potential of the stem cells. Additionally,in vitro transduced cells must be tested for sterility, toxicity, etc.before transplantation. Prior to transplanting into recipients, the stemcells often have to undergo conditioning with chemotherapy or radiationto ensure engraftment. The process usually requires hospitalization ofpatients for at least several days and sometimes longer. Overall, thisis an arduous, expensive and high risk procedure that greatly limits theutility of stem cell gene therapy. A procedure is needed that offers abetter alternative to current stem cell transduction methods without theneed for purification and ex vivo transduction.

SUMMARY

Provided herein are adeno-associated virus (AAV) vectors (e.g., Clade Fvectors such as a replication-defective adeno-associated virus (AAV)comprising a correction genome enclosed in a capsid, the capsid being anAAV Clade F capsid) for editing the genome of a cell via homologousrecombination and methods of use and kits thereof.

In some aspects, the disclosure provides a replication-defectiveadeno-associated virus (AAV) comprising a correction genome enclosed ina capsid, the capsid being an AAV Clade F capsid; and the correctiongenome comprising (a) an editing element selected from aninternucleotide bond or a nucleotide sequence for integration into atarget locus of a mammalian chromosome, (b) a 5′ homologous armnucleotide sequence 5′ of the editing element, having homology to a 5′region of the mammalian chromosome relative to the target locus, and (c)a 3′ homologous arm nucleotide sequence 3′ of the editing element,having homology to a 3′ region of the mammalian chromosome relative tothe target locus. In some aspects, the also disclosure providesreplication-defective adeno-associated virus (AAV) comprising acorrection genome enclosed in a capsid, the capsid being an AAV Clade Fcapsid; and the correction genome comprising an editing elementnucleotide sequence for integration into a target locus of a mammalianchromosome, the correction genome having an essential absence of apromoter operatively linked to the editing element nucleotide sequence.In further aspects, the disclosure provides a replication-defectiveadeno-associated virus (AAV) comprising a correction genome enclosed ina capsid, wherein the capsid being an AAV Clade F capsid; the correctiongenome comprising an editing element selected from an internucleotidebond or a nucleotide sequence for integration into a target locus of amammalian chromosome in a cell; and the AAV having a chromosomalintegration efficiency of at least about 1% for integrating the editingelement into the target locus of the mammalian chromosome in the cell.Other aspects of the disclosure relate to a gene editing vectorcomprising a replication-defective adeno-associated virus (AAV)comprising a correction genome enclosed in an AAV capsid, the correctiongenome comprising an editing element selected from an internucleotidebond or a nucleotide sequence for integration into a target locus of amammalian cell chromosome; a 5′ homologous arm nucleotide sequence 5′ ofthe editing element having homology to a 5′ region of the chromosomerelative to the target locus; a 3′ homologous arm nucleotide sequence 3′of the editing element having homology to a 3′ region of the chromosomerelative to the target locus; and wherein the AAV has a chromosomalintegration efficiency of at least 10% for integrating the editingelement into the target locus of the mammalian cell chromosome in theabsence of an exogenous nuclease.

In some embodiments of any one of the AAVs provided herein, the AAV hasa chromosomal integration efficiency of at least about 1% in the absenceof an exogenous nuclease for integrating the editing element into thetarget locus of the mammalian chromosome in the cell.

In some embodiments of any one of the AAVs provided herein, thecorrection genome comprises a 5′ inverted terminal repeat (5′ ITR)nucleotide sequence 5′ of the 5′ homologous arm nucleotide sequence, anda 3′ inverted terminal repeat (3′ ITR) nucleotide sequence 3′ of the 3′homologous arm nucleotide sequence. In some embodiments, the 5′ ITRnucleotide sequence and the 3′ ITR nucleotide sequence are substantiallyidentical to an AAV2 virus 5′ITR and an AAV2 virus 3′ ITR, respectively.In some embodiments, the 5′ ITR nucleotide sequence and the 3′ ITRnucleotide sequence are substantially mirror images of each other. Insome embodiments, the 5′ ITR nucleotide sequence has at least 95%sequence identity to SEQ ID NO:36, and the 3′ ITR nucleotide sequencehas at least 95% sequence identity to SEQ ID NO:37. In some embodiments,the 5′ ITR nucleotide sequence and the 3′ ITR nucleotide sequence aresubstantially identical to an AAV5 virus 5′ITR and an AAV5 virus 3′ ITR,respectively. In some embodiments, the 5′ ITR nucleotide sequence andthe 3′ ITR nucleotide sequence are substantially mirror images of eachother. In some embodiments, the 5′ ITR nucleotide sequence has at least95% sequence identity to SEQ ID NO:38, and the 3′ ITR nucleotidesequence has at least 95% sequence identity to SEQ ID NO:39.

In some embodiments of any one of the AAVs provided herein, thecorrection genome has an essential absence of a promoter operativelylinked to the editing element nucleotide sequence. In some embodimentsof any one of the AAVs provided herein, the correction genome furthercomprises an exogenous promoter operatively linked to the editingelement.

In some embodiments of any one of the AAVs provided herein, thereplication-defective AAV genome comprises an essential absence of anAAV rep gene and an AAV cap gene.

In some embodiments of any one of the AAVs provided herein, each of the5′ and 3′ homologous arm nucleotide sequences independently has anucleotide length of between about 500 to 1000 nucleotides or betweenabout 600 to 1000 nucleotides. In some embodiments, the 5′ and 3′homologous arm nucleotide sequences have substantially equal nucleotidelengths. In some embodiments, the 5′ and 3′ homologous arm nucleotidesequences have asymmetrical nucleotide lengths. In some embodiments, the5′ homologous arm nucleotide sequence has at least about 95% nucleotidesequence identity to the 5′ region of the mammalian chromosome relativeto the target locus. In some embodiments, the 3′ homologous armnucleotide sequence has at least about 95% nucleotide sequence identityto the 3′ region of the mammalian chromosome relative to the targetlocus. In some embodiments, the 5′ homologous arm nucleotide sequencehas 100% sequence identity to the 5′ region of the mammalian chromosomerelative to the target locus and the 3′ homologous arm nucleotidesequence has 100% sequence identity to the 3′ region of the mammalianchromosome relative to the target locus.

In some embodiments of any one of the AAVs provided herein, the editingelement consists of one nucleotide. In some embodiments, the targetlocus is a nucleotide sequence consisting of one nucleotide, and thetarget locus represents a point mutation of the mammalian chromosome.

In some embodiments, the target locus can comprise an intron of amammalian chromosome. In some embodiments, the target locus can comprisean exon of a mammalian chromosome. In some embodiments, the target locuscan comprise a non-coding region of a mammalian chromosome. In someembodiments, the target locus can comprise a regulatory region of amammalian chromosome. In some embodiments, the target locus may be alocus associated with a disease state as described herein.

In some embodiments of any one of the AAVs provided herein, the editingelement comprises at least 1, 2, 10, 100, 200, 500, 1000, 1500, 2000,3000, 4000, or 5000 nucleotides. In some embodiments, the editingelement comprises 1 to 5500, 1 to 5000, 1 to 4500, 1 to 4000, 1 to 3000,1 to 2000, 1 to 1000, 1 to 500, 1 to 200, or 1 to 100 nucleotides, or 2to 5500, 2 to 5000, 2 to 4500, 2 to 4000, 2 to 3000, 2 to 2000, 2 to1000, 2 to 500, 2 to 200, or 2 to 100 nucleotides, or 10 to 5500, 10 to5000, 10 to 4500, 10 to 4000, 10 to 3000, 10 to 2000, 10 to 1000, 10 to500, 10 to 200, or 10 to 100 nucleotides.

In some embodiments of any one of the AAVs provided herein, the editingelement comprises an exon, an intron, a 5′ untranslated region (UTR), a3′ UTR, a promoter, a splice donor, a splice acceptor, a sequenceencoding or non-coding RNA, an insulator, a gene, or a combinationthereof. In some embodiments of any one of the AAVs provided herein, theediting element is a fragment of a coding sequence of a gene within orspanning the target locus.

In some embodiments of any one of the AAVs provided herein, the targetlocus is a nucleotide sequence comprising n nucleotides where n is aninteger greater than or equal to one; the editing element comprises mnucleotides where m is an integer equal to n; and the editing elementrepresents a substitution for the target locus of the mammalianchromosome. In some embodiments of any one of the AAVs provided herein,the target locus is a nucleotide sequence comprising n nucleotides wheren is an integer greater than or equal to one; the editing elementcomprises m nucleotides where m is an integer greater than n; and theediting element represents a substitutive addition for the target locusof the mammalian chromosome. In some embodiments of any one of the AAVsprovided herein, the target locus is a nucleotide sequence comprising nnucleotides where n is an integer greater than or equal to two; theediting element comprises m nucleotides where m is an integer less thann; and the editing element represents a substitutive deletion for thetarget locus of the mammalian chromosome. In some embodiments of any oneof the AAVs provided herein, the target locus is an internucleotidebond; the editing element comprises m nucleotides where m is an integergreater than or equal to one; and the editing element represents anaddition for the target locus of the mammalian chromosome.

In some embodiments of any one of the AAVs provided herein, the editingelement is an internucleotide bond. In some embodiments, the targetlocus is a nucleotide sequence comprising one or more nucleotides, andthe editing element comprises a deletion for the target locus of themammalian chromosome.

In some embodiments of any one of the AAVs provided herein, the targetlocus of the mammalian chromosome is a mutant target locus comprisingone or more mutant nucleotides, relative to a corresponding wild typemammalian chromosome. In some embodiments, the mutant target locuscomprises a point mutation, a missense mutation, a nonsense mutation, aninsertion of one or more nucleotides, a deletion of one or morenucleotides, or combinations thereof. In some embodiments, the mutanttarget locus comprises an amorphic mutation, a neomorphic mutation, oran antimorphic mutation. In some embodiments, the mutant target locuscomprises an autosomal dominant mutation, an autosomal recessivemutation, a heterozygous mutation, a homozygous mutation, orcombinations thereof. In some embodiments, the mutant target locus isselected from a promoter, an enhancer, a signal sequence, an intron, anexon, a splice donor site, a splice acceptor site, an internal ribosomeentry site, an inverted exon, an insulator, a gene, a chromosomalinversion, and a chromosomal translocation within the mammalianchromosome.

In some embodiments of any one of the AAVs provided herein, the AAVClade F capsid comprises at least one protein selected from Clade F VP1,Clade F VP2 and Clade F VP3. In some embodiments, the AAV Clade F capsidcomprises at least two proteins selected from Clade F VP1, Clade F VP2and Clade F VP3. In some embodiments, the AAV Clade F capsid comprisesClade F VP1, Clade F VP2 and Clade F VP3 proteins. In some embodiments,the AAV Clade F capsid comprises a VP1, VP2, or VP3 protein that has atleast 90% amino acid sequence identity to amino acids 1 to 736, aminoacids 138 to 736 or amino acids 203 to 736 of SEQ ID NO:1, respectively,which correspond to the amino acid sequences of AAV9 capsid proteinsVP1, VP2 and VP3, respectively. In some embodiments, the AAV Clade Fcapsid comprises VP1 and VP2 proteins that have at least 90% amino acidsequence identity to amino acids 1 to 736 and amino acids 138 to 736 ofSEQ ID NO:1, respectively, which correspond to the amino acid sequencesof AAV9 capsid proteins VP1 and VP2, respectively; VP1 and VP3 proteinsthat have at least 90% amino acid sequence identity to amino acids 1 to736 and amino acids 203 to 736 of SEQ ID NO:1, respectively, whichcorrespond to the amino acid sequences of AAV9 capsid proteins VP1 andVP3, respectively; or VP2 and VP3 proteins that have at least 90% aminoacid sequence identity to amino acids 138 to 736 and amino acids 203 to736 of SEQ ID NO:1, respectively, which correspond to the amino acidsequences of AAV9 capsid proteins VP2 and VP3, respectively. In someembodiments, the AAV Clade F capsid comprises VP1, VP2, and VP3 proteinsthat have at least 90% amino acid sequence identity to amino acids 1 to736, amino acids 138 to 736 and amino acids 203 to 736 of SEQ ID NO:1,respectively, which correspond to the amino acid sequences of AAV9capsid proteins VP1, VP2 and VP3, respectively. In some embodiments, theAAV Clade F capsid comprises a VP1, VP2, or VP3 protein that has atleast 90% amino acid sequence identity to amino acids 1 to 736, aminoacids 138 to 736 or amino acids 203 to 736 of any one of SEQ ID NOs: 2,3, 5, 6, 11, 7, 8, 9, 10, 4, 12, 14, 15, 16, 17 or 13, respectively,which correspond to the amino acid sequences of AAVF1 through AAVF9 andAAVF11 through AAVF17 capsid proteins VP1, VP2 and VP3, respectively. Insome embodiments, the AAV Clade F capsid comprises VP1 and VP2 proteinsthat have at least 90% amino acid sequence identity to amino acids 1 to736 and amino acids 138 to 736 of any one of SEQ ID NOs: 2, 3, 5, 6, 11,7, 8, 9, 10, 4, 12, 14, 15, 16, 17 or 13, respectively, which correspondto the amino acid sequences of AAVF1 through AAVF9 and AAVF11 throughAAVF17 capsid proteins VP1 and VP2, respectively; VP1 and VP3 proteinsthat have at least 90% amino acid sequence identity to amino acids 1 to736 and amino acids 203 to 736 of any one of SEQ ID NOs: 2, 3, 5, 6, 11,7, 8, 9, 10, 4, 12, 14, 15, 16, 17 or 13, respectively, which correspondto the amino acid sequences of AAVF1 through AAVF9 and AAVF11 throughAAVF17 capsid proteins VP1 and VP3, respectively; or VP2 and VP3proteins that have at least 90% amino acid sequence identity to aminoacids 138 to 736 and amino acids 203 to 736 of any one of SEQ ID NOs: 2,3, 5, 6, 11, 7, 8, 9, 10, 4, 12, 14, 15, 16, 17 or 13, respectively,which correspond to the amino acid sequences of AAVF1 through AAVF9 andAAVF11 through AAVF17 capsid proteins VP2 and VP3, respectively. In someembodiments, the AAV Clade F capsid comprises VP1, VP2, and VP3 proteinsthat have at least 90% amino acid sequence identity to amino acids 1 to736, amino acids 138 to 736 and amino acids 203 to 736 of any one of SEQID NOs: 2, 3, 5, 6, 11, 7, 8, 9, 10, 4, 12, 14, 15, 16, 17 or 13,respectively, which correspond to the amino acid sequences of AAVF1through AAVF9 and AAVF11 through AAVF17 capsid proteins VP1, VP2 andVP3, respectively. In some embodiments, the AAV Clade F capsid comprisesa VP1, VP2, or VP3 protein that is encoded by a nucleotide sequencecomprising at least 90% nucleotide sequence identity to SEQ ID NO: 18,respectively, which corresponds to a nucleotide sequence encoding AAV9capsid proteins VP1, VP2 and VP3, respectively. In some embodiments, theAAV Clade F capsid comprises VP1 and VP2 proteins that are encoded bynucleotide sequences comprising at least 90% nucleotide sequenceidentity to SEQ ID NOs:18; VP1 and VP3 proteins that are encoded by anucleotide sequence comprising at least 90% nucleotide sequence identityto SEQ ID NOs: 18; or VP2 and VP3 proteins that are encoded by anucleotide sequence comprising at least 90% nucleotide sequence identityto SEQ ID NOs:18. In some embodiments, the AAV Clade F capsid comprisesVP1, VP2, and VP3 proteins that are encoded by a nucleotide sequencecomprising at least 90% nucleotide sequence identity to SEQ ID NO: 18,which corresponds to a nucleotide sequence encoding AAV9 capsid proteinsVP1, VP2 and VP3. In some embodiments, the AAV Clade F capsid comprisesa VP1, VP2, or VP3 protein that is encoded by a nucleotide sequencecomprising at least 90% nucleotide sequence identity to any one of SEQID NOs: 20, 21, 22, 23, 25, 24, 27, 28, 29, 26, 30, 31, 32, 33, 34 or35, respectively, which correspond to nucleotide sequences encodingAAVF1 through AAVF9 and AAVF11 through AAVF17 capsid proteins VP1, VP2and VP3, respectively. In some embodiments, the AAV Clade F capsidcomprises VP1 and VP2 proteins that are encoded by nucleotide sequencescomprising at least 90% nucleotide sequence identity to any one of SEQID NOs:20-35; VP1 and VP3 proteins that are encoded by a nucleotidesequence comprising at least 90% nucleotide sequence identity to any oneof SEQ ID NOs:20-35; or VP2 and VP3 proteins that are encoded by anucleotide sequence comprising at least 90% nucleotide sequence identityto any one of SEQ ID NOs:20-35. In some embodiments, the AAV Clade Fcapsid comprises VP1, VP2, and VP3 proteins that are encoded by anucleotide sequence comprising at least 90% nucleotide sequence identityto any one of SEQ ID NOs: 20, 21, 22, 23, 25, 24, 27, 28, 29, 26, 30,31, 32, 33, 34 or 35, which correspond to nucleotide sequences encodingAAVF1 through AAVF9 and AAVF11 through AAVF17 capsid proteins VP1, VP2and VP3, respectively. In some embodiments, the AAV Clade F capsidcomprises AAV9 VP1, VP2, or VP3 capsid proteins, which correspond toamino acids 1 to 736, amino acids 138 to 736 and amino acids 203 to 736as set forth in SEQ ID NO:1, respectively. In some embodiments, the AAVClade F capsid comprises AAV9 VP1 and VP2 capsid proteins, whichcorrespond to amino acids 1 to 736 and amino acids 138 to 736 as setforth in SEQ ID NO:1, respectively; AAV9 VP1 and VP3 capsid proteins,which correspond to amino acids 1 to 736 and amino acids 203 to 736 asset forth in SEQ ID NO: 1, respectively; or AAV9 VP2 and VP3 capsidproteins, which correspond to amino acids 138 to 736 and amino acids 203to 736 as set forth in SEQ ID NO:1, respectively. In some embodiments,the AAV Clade F capsid comprises AAV9 capsid proteins VP1, VP2 and VP3,which correspond to amino acids 1 to 736, amino acids 138 to 736 andamino acids 203 to 736 as set forth in SEQ ID NO:1, respectively. Insome embodiments, the AAV Clade F capsid comprises a VP1 capsid proteinselected from a VP1 capsid protein of any one of AAVF1 through AAVF9 andAAVF11 through AAVF17, which corresponds to amino acids 1 to 736 as setforth in SEQ ID NOs: 2, 3, 5, 6, 11, 7, 8, 9, 10, 4, 12, 14, 15, 16, 17or 13, respectively. In some embodiments, the AAV Clade F capsidcomprises a VP1 and a VP2 capsid protein independently selected from aVP1 and VP2 capsid protein of any one of AAVF1 through AAVF9 and AAVF11through AAVF17, which correspond to amino acids 1 to 736 and amino acids138 to 736 as set forth in SEQ ID NOs: 2, 3, 5, 6, 11, 7, 8, 9, 10, 4,12, 14, 15, 16, 17 or 13, respectively. In some embodiments, the AAVClade F capsid comprises a VP1, a VP2 and a VP3 capsid proteinindependently selected from a VP1, VP2 and VP3 capsid protein of any oneof AAVF1 through AAVF9 and AAVF11 through AAVF17, which correspond toamino acids 1 to 736, amino acids 138 to 736 and amino acids 203 to 736as set forth in SEQ ID NOs: 2, 3, 5, 6, 11, 7, 8, 9, 10, 4, 12, 14, 15,16, 17 or 13, respectively. In some embodiments, the AAV Clade F capsidcomprises each of the VP1, VP2 and VP3 capsid proteins of any one ofAAVF1 through AAVF9 and AAVF11 through AAVF17, which correspond to aminoacids 1 to 736, amino acids 138 to 736 and amino acids 203 to 736 as setforth in SEQ ID NOs: 2, 3, 5, 6, 11, 7, 8, 9, 10, 4, 12, 14, 15, 16, 17or 13, respectively.

In some embodiments of any one of the AAVs provided, the Clade F capsidscomprises a polypeptide sequence having a percent sequence identity ofat least 95% to a polypeptide sequence selected from the group of AAVF5(SEQ ID NO: 11), AAVF7 (SEQ ID NO: 8), AAVF12 (SEQ ID NO: 12), AAVF15(SEQ ID NO: 16), AAVF17 (SEQ ID NO: 13), AAVF9 (SEQ ID NO: 10), AAVF16(SEQ ID NO: 17), variants, fragments, mutants and any combinationthereof. As used herein, AAVF1, AAVF2, AAVF3, AAVF4, AAVF5, AAVF6,AAVF7, AAVF8, AAVF9, AAVF10, AAVF11, AAVF12, AAVF13, AAVF14, AAVF15,AAVF16, and AAVF17 are also referred to as AAVHSC1, AAVHSC2, AAVHSC3,AAVHSC4, AAVHSC5, AAVHSC6, AAVHSC7, AAVHSC8, AAVHSC9, AAVHSC10,AAVHSC11, AAVHSC12, AAVHSC13, AAVHSC14, AAVHSC15, AAVHSC16, andAAVHSC17, respectively. In other words, any recitation of AAVF1, AAVF2,AAVF3, AAVF4, AAVF5, AAVF6 AAVF7, AAVF8, AAVF9, AAVF10, AAVF11, AAVF12,AAVF13, AAVF14, AAVF15, AAVF16, or AAVF17 is equivalent to and may bereplaced with AAVHSC1, AAVHSC2, AAVHSC3, AAVHSC4, AAVHSC5, AAVHSC6,AAVHSC7, AAVHSC8, AAVHSC9, AAVHSC10, AAVHSC11, AAVHSC12, AAVHSC13,AAVHSC14, AAVHSC15, AAVHSC16, or AAVHSC17, respectively.

In some embodiments of any one of the AAVs provided herein, themammalian chromosome is selected from human chromosome 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, X and Y. Insome embodiments of any one of the AAVs provided herein, the mammalianchromosome is selected from mouse chromosome 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, X and Y. In some embodiments,the mammalian chromosome is not human chromosome 19.

In some embodiments of any one of the AAVs provided herein, themammalian chromosome is a somatic cell chromosome. In some embodiments,the somatic cell is from a tissue selected from the group consisting ofconnective tissue (including blood), muscle tissue, nervous tissue, andepithelial tissue. In some embodiments, the somatic cell is from anorgan selected from the group consisting of lung, heart, liver, kidney,muscle, brain, eye, breast, bone, and cartilage. In some embodiments,the somatic cell is a CD34+ cell.

In some embodiments of any one of the AAVs provided herein, the cell isa stem cell. In some embodiments, the stem cell is a hematopoietic stemcell, a cord blood stem cell, a bone marrow stem cell, a fetal liverstem cell, or a peripheral blood stem cell. In some embodiments, thecell is selected from the group consisting of a CD34+ Hematopoietic stemcell line (HSC), a K562 CD34+ leukemia cell line, a HepG2 human livercell line, a peripheral blood stem cell, a cord blood stem cell, a CD34+peripheral blood stem cell, a WI-38 human diploid fibroblast cell line,a MCF7 human breast cancer cell line, a Y79 human retinoblastoma cellline, a SCID-X1 LBL human EBV-immortalized B cell line, a primary humanhepatocyte, a primary hepatic sinusoidal endothelial cell, and a primaryskeletal muscle myoblast.

In some embodiments of any one of the AAVs provided herein, the AAV hasa chromosomal integration efficiency of at least about 5% forintegrating the editing element into the target locus of the mammalianchromosome in the cell. In some embodiments, the AAV has a chromosomalintegration efficiency of at least about 10% for integrating the editingelement into the target locus of the mammalian chromosome in the cell.

Other aspects of the disclosure relate to a composition comprising anAAV as described herein, wherein the composition is in apharmaceutically acceptable formulation. In some embodiments, theformulation is constituted for administration to a mammal. In someembodiments, the formulation is constituted for administration to amammal via intravenous injection, subcutaneous injection, intramuscularinjection, autologous cell transfer, or allogeneic cell transfer. Insome embodiments, the pharmaceutically acceptable formulation comprisesan excipient. In some embodiments, the excipient is selected from acarrier, an adjuvant and a vehicle, or combinations thereof.

Yet other aspects of the disclosure relate to a packaging system forrecombinant preparation of an adeno-associated virus (AAV), wherein thepackaging system comprises a Rep nucleotide sequence encoding one ormore AAV Rep proteins; a Cap nucleotide sequence encoding one or moreAAV Cap proteins of an AAV Clade F capsid; and a correction genome asdescribed herein; wherein the packaging system is operative in a cellfor enclosing the correction genome in the capsid to form theadeno-associated virus. In some embodiments, the packaging systemcomprises a first vector comprising the Rep nucleotide sequence and theCap nucleotide sequence, and a second vector comprising the correctiongenome. In some embodiments, the AAV Clade F capsid comprises at leastone protein selected from Clade F VP1, Clade F VP2 and Clade F VP3. Insome embodiments, the AAV Clade F capsid comprises at least two proteinsselected from Clade F VP1, Clade F VP2 and Clade F VP3. In someembodiments, the AAV Clade F capsid comprises Clade F VP1, Clade F VP2and Clade F VP3 proteins. In some embodiments, the AAV Clade F capsid isany AAV Clade F capsid as described herein. In some embodiments, the Repnucleotide sequence encodes an AAV2 Rep protein. In some embodiments,the AAV2 Rep protein encoded is at least one of Rep 78/68 or Rep 68/52.In some embodiments, the nucleotide sequence encoding the AAV2 Repprotein comprises a nucleotide sequence having a minimum percentsequence identity to the AAV2 Rep nucleotide sequence of SEQ ID NO:40,wherein the minimum percent sequence identity is at least 70% across thelength of the nucleotide sequence encoding the AAV2 Rep protein. In someembodiments of any one of the packaging systems provided, the packagingsystem further comprises a third vector, wherein the third vector is ahelper virus vector. In some embodiments, the helper virus vector is anindependent third vector. In some embodiments, the helper virus vectoris integral with the first vector. In some embodiments, the helper virusvector is integral with the second vector. In some embodiments, thethird vector comprises genes encoding helper virus proteins. In someembodiments, the helper virus is selected from the group consisting ofadenovirus, herpes virus (including herpes simplex virus (HSV)),vaccinia virus, and cytomegalovirus (CMV). In some embodiments, thehelper virus is adenovirus. In some embodiments, the adenovirus genomecomprises one or more adenovirus RNA genes selected from the groupconsisting of E1, E2, E4 and VA. In some embodiments, the helper virusis HSV. In some embodiments, the HSV genome comprises one or more of HSVgenes selected from the group consisting of UL5/8/52, ICPO, ICP4, ICP22and UL30/UL42. In some embodiments, the first vector and the thirdvector are contained within a first transfecting plasmid. In someembodiments, the nucleotides of the second vector and the third vectorare contained within a second transfecting plasmid. In some embodiments,the nucleotides of the first vector and the third vector are cloned intoa recombinant helper virus. In some embodiments, the nucleotides of thesecond vector and the third vector are cloned into a recombinant helpervirus. In some embodiments, the AAV capsid is the capsid of a Clade FAAV selected from the group consisting of AAV9, AAVF1, AAVF2, AAVF3,AAVF4, AAVF5, AAVF6, AAVF7, AAVF8, AAVF9, AAVF11, AAVF12, AAVF13,AAVF14, AAVF15, AAVF16, AAVF17, AAVHU31, and AAVHU32. In someembodiments, any of the packaging systems described herein are comprisedwithin a kit.

Other aspects of the disclosure relate to a method for recombinantpreparation of an adeno-associated virus (AAV), wherein the methodcomprises transfecting or transducing a cell with a packaging system asdescribed herein under conditions operative for enclosing the correctiongenome in the capsid to form the AAV.

In other aspects, the disclosure provides a method for editing a targetlocus of a mammalian genome, wherein the method comprises transducing acell comprising the mammalian genome with an adeno-associated virus(AAV) as described herein. In some embodiments, the cell is a mammalianstem cell. In some embodiments, the mammalian cell is from a tissueselected from the group consisting of connective tissue (includingblood), muscle tissue, nervous tissue, and epithelial tissue. In someembodiments, the mammalian cell is from an organ selected from the groupconsisting of lung, heart, liver, kidney, muscle, brain, eye, breast,bone, and cartilage. In some embodiments, the mammalian cell is a stemcell. In some embodiments, the stem cell is a hematopoietic stem cell, acord blood stem cell, or peripheral blood stem cell. In someembodiments, the mammalian cell is a myoblast, an endothelial cell, aliver cell, a fibroblast, a breast cell, a lymphocyte, or a retinalcell. Other aspects of the disclosure relate to a cell obtainable by anymethod described herein.

Another aspect of the disclosure relates to a method for editing atarget locus of a mammalian genome, wherein the method comprises: (a)obtaining mammalian cells from a mammal; (b) culturing the mammaliancells ex-vivo to form an ex-vivo culture; (c) transducing the mammaliancells with an adeno-associated virus (AAV) as described herein in theex-vivo culture to form transduced mammalian cells; and (d)administering the transduced mammalian cells to the mammal.

In other aspects, the disclosure provides a method for editing a targetlocus of a mammalian genome, wherein the method comprises: (a) obtainingmammalian cells from a first mammal; (b) culturing the mammalian cellsex-vivo to form an ex-vivo culture; (c) transducing the mammalian cellswith an adeno-associated virus (AAV) as described herein in the ex-vivoculture to form transduced mammalian cells; and (d) administering thetransduced mammalian cells to a second mammal. In some embodiments, thefirst mammal and the second mammal are the same species. In someembodiments, the mammalian cells are from a tissue selected from thegroup consisting of connective tissue (including blood), muscle tissue,nervous tissue, and epithelial tissue. In some embodiments, themammalian cells are from an organ selected from the group consisting oflung, heart, liver, kidney, muscle, brain, eye, breast, bone, andcartilage. In some embodiments, the mammalian cells are stem cells. Insome embodiments, the stem cells are hematopoietic stem cells, cordblood stem cells, or peripheral blood stem cells. In some embodiments,the mammalian cells are a CD34+ cells. In some embodiments, themammalian cells are myoblasts, endothelial cells, liver cells,fibroblasts, breast cells, lymphocytes, or retinal cells.

Another aspect of the disclosure provides a method for editing a targetlocus of a mammalian genome, wherein the method comprises administeringan AAV as described herein or a composition as described herein to amammal in an amount effective to transduce cells of the mammal with theAAV in-vivo.

In some embodiments of any one of the methods provided, the AAV istransduced or administered without co-transducing or co-administering anexogenous nuclease or a nucleotide sequence that encodes an exogenousnuclease.

In some embodiments of any one of the methods provided, the AAV has achromosomal integration efficiency of at least about 1% for integratingthe editing element into the target locus of the mammalian chromosome.In some embodiments, the chromosomal integration efficiency of the AAVis at least about 2%, 3%, 4% or 5% for integrating the editing elementinto the target locus of the mammalian chromosome. In some embodiments,the editing element of the correction genome is integrated into thetarget locus of the mammalian chromosome with a chromosomal integrationefficiency of at least 10%, 20%, 40%, or 50% of the mammalian cells. Insome embodiments, the editing element of the correction genome isintegrated into the target locus of the mammalian chromosome with achromosomal integration efficiency ranging from 10% to 70%, 20% to 70%,40% to 70%, or 50% to 70% of the mammalian cells.

In some embodiments of any one of the methods provided, the AAV has achromosomal integration efficiency further characterized by an allelefrequency in a population of cells of at least about 10% for the allelecomprising the editing element integrated into the target locus of themammalian chromosome. In some embodiments, the AAV has a chromosomalintegration efficiency further characterized by an allele frequency in apopulation of cells of at least about 50% for the allele comprising theediting element integrated into the target locus of the mammalianchromosome. In some embodiments, the AAV has a chromosomal integrationefficiency further characterized by an allele frequency in a populationof cells of at least about 75% for the allele comprising the editingelement integrated into the target locus of the mammalian chromosome. Insome embodiments, the allele frequency in a population of cells is anallele frequency in a population of cells in vitro.

Other aspects of the disclosure relate to a method for generating atransgenic non-human animal, the method comprising administering an AAVas described herein or a composition as described herein to a non-humananimal; or transducing a non-human animal cell with the AAV as describedherein or the composition as described herein and implanting the cellinto a host non-human animal under conditions sufficient to generate atransgenic non-human animal from the host non-human animal (e.g., byallowing the implanted cell to form or become part of an embryo, whichthen develops in the host into a transgenic non-human animal). In someembodiments, the transgenic non-human animal is crossed with anothernon-human animal to generate further transgenic non-human animals. Insome embodiments, the non-human animal cell is derived from a zygote oran embryo of a non-human animal. In some embodiments, the non-humananimal is a mouse, rat, rabbit, pig, bovine, sheep, goat, chicken, cat,dog, ferret, or primate.

Other aspects of the disclosure relate to a transgenic non-human animalobtainable by a method described herein, such as a method describedabove. In some embodiments, the transgenic non-human animal is a mouse,rat, rabbit, pig, bovine, sheep, goat, chicken, cat, dog, ferret, orprimate.

Yet other aspects of the disclosure relate to tissue derived from atransgenic non-human animal as described herein. In some embodiments,the tissue is selected from the group consisting of connective tissue(including blood), muscle tissue, nervous tissue, endothelial tissue andepithelial tissue. In some embodiments, the tissue is from an organselected from the group consisting of lung, heart, liver, kidney,muscle, brain, eye, breast, bone, and cartilage.

Other aspects of the disclosure relate to a cell derived from atransgenic non-human animal as described herein. In some embodiments,the cell is a primary cell. In some embodiments, the cell is a CD34+cell, a myoblast, an endothelial cell, a liver cell, a fibroblast, abreast cell, a lymphocyte, or a retinal cell. In some embodiments, thecell is an inducible pluripotent stem (iPS) cell. In some embodiments,the cell is from a tissue selected from the group consisting ofconnective tissue (including blood), muscle tissue, nervous tissue,endothelial tissue, and epithelial tissue. In some embodiments, the cellis from an organ selected from the group consisting of lung, heart,liver, kidney, muscle, brain, eye, breast, bone, and cartilage. In someembodiments, the cell is a stem cell. In some embodiments, the stem cellis a hematopoietic stem cell, a cord blood stem cell, or peripheralblood stem cell.

According to certain embodiments, adeno-associated virus (AAV) Clade Fvectors (e.g., replication-defective AAVs comprising correction genomesenclosed in a Clade F capsid) or AAV vector variants (e.g.,replication-defective AAVs comprising capsid variants relative to AAV9capsids) for editing the genome of a cell are provided. In certainembodiments, an AAV Clade F vector or AAV vector variant may compriseone or more Clade F capsids or one or more capsid variants (relative toan AAV9 capsid), an editing element (also referred to herein as atargeting cassette) comprising one or more therapeutic nucleotidesequences to be integrated into a target locus (also referred to hereinas a target site) of the genome, a 5′ homologous arm polynucleotidesequence flanking the editing element (targeting cassette) and havinghomology to a region that is upstream of the target locus (target site),and a 3′ homologous arm polynucleotide sequence flanking the editingelement (targeting cassette) and having homology to a region that isdownstream of the target locus (target site). The editing element(target cassette) may be contained within a correction genome asdescribed herein comprising inverted terminal repeats (ITRs) asdescribed herein. In certain embodiments, the one or more Clade Fcapsids or capsid variants may be any of the Clade F capsids or capsidvariants described herein. In certain embodiments, the one or more CladeF capsids or capsid variants may comprise a polypeptide sequenceselected from the group of AAVF7 (SEQ ID NO: 8), AAVF12 (SEQ ID NO: 12),AAVF15 (SEQ ID NO: 16), AAVF17 (SEQ ID NO: 13), variants, fragments,mutants and any combination thereof. In certain embodiments, the one ormore Clade F capsids or the one or more capsid variants may comprise apolypeptide sequence selected from the group of AAVF5 (SEQ ID NO: 11),AAVF7 (SEQ ID NO: 8), AAVF12 (SEQ ID NO: 12), AAVF15 (SEQ ID NO: 16),AAVF17 (SEQ ID NO: 13), AAVF9 (SEQ ID NO: 10), AAVF16 (SEQ ID NO: 17),variants, fragments, mutants and any combination thereof. In certainembodiments, the one or more Clade F capsids or capsid variants maycomprise a polypeptide sequence having a percent sequence identity of atleast 95% to a polypeptide sequence selected from the group of AAVF7(SEQ ID NO: 8), AAVF12 (SEQ ID NO: 12), AAVF15 (SEQ ID NO: 16), AAVF17(SEQ ID NO: 13), variants, fragments, mutants and any combinationthereof. In certain embodiments, the one or more Clade F capsids orcapsid variants may comprise a polypeptide sequence having a percentsequence identity of at least 95% to a polypeptide sequence selectedfrom the group of AAVF5 (SEQ ID NO: 11), AAVF7 (SEQ ID NO: 8), AAVF12(SEQ ID NO: 12), AAVF15 (SEQ ID NO: 16), AAVF17 (SEQ ID NO: 13), AAVF9(SEQ ID NO: 10), AAVF16 (SEQ ID NO: 17), variants, fragments, mutantsand any combination thereof. In certain embodiments, the target locus(target site) may be a safe harbor site. In certain embodiments, thesafe harbor site may be the AAVS1 locus on chromosome 19. In certainembodiments, the target locus (target site) may be a locus associatedwith a disease state as described herein. In certain embodiments, thecell may be a stem cell. In certain embodiments, the stem cell may be ahematopoietic stem cell, a pluripotent stem cell, an embryonic stemcell, or a mesenchymal stem cell.

According to certain embodiments, methods of editing the genome of acell are provided. In certain embodiments, the methods of editing thegenome of a cell may comprise transducing the cell with one or more AAVClade F vectors (e.g., replication-defective AAVs comprising correctiongenomes enclosed in a Clade F capsid) or AAV vector variants (e.g.,replication-defective AAVs comprising capsid variants relative to AAV9capsids) as described herein. In certain embodiments, the transductionmay be performed without additional exogenous nucleases. In certainembodiments, AAV Clade F vectors or AAV vector variants may comprise oneor more Clade F capsids or capsid variants (relative to an AAV9 capsid),an editing element (targeting cassette) comprising one or moretherapeutic nucleotide sequences to be integrated into a target locus(target site) of the genome, a 5′ homologous arm polynucleotide sequenceflanking the editing element (targeting cassette) and having homology toa region that is upstream of the target locus (target site), and a 3′homologous arm polynucleotide sequence flanking the editing element(targeting cassette) and having homology to a region that is downstreamof the target locus (target site). The editing element (target cassette)may be contained within a correction genome as described hereincomprising ITRs as described herein. In certain embodiments, the one ormore Clade F capsids or capsid variants may comprise a polypeptidesequence selected from the group of AAVF7 (SEQ ID NO: 8), AAVF12 (SEQ IDNO: 12), AAVF15 (SEQ ID NO: 16), AAVF17 (SEQ ID NO: 13), variants,fragments, mutants and any combination thereof. In certain embodiments,the one or more Clade F capsids or capsid variants may comprise apolypeptide sequence selected from the group of AAVF5 (SEQ ID NO: 11),AAVF7 (SEQ ID NO: 8), AAVF12 (SEQ ID NO: 12), AAVF15 (SEQ ID NO: 16),AAVF17 (SEQ ID NO: 13), AAVF9 (SEQ ID NO: 10), AAVF16 (SEQ ID NO: 17),variants, fragments, mutants and any combination thereof. In certainembodiments, the one or more Clade F capsids or capsid variants maycomprise a polypeptide sequence having a percent sequence identity of atleast 95% to a polypeptide sequence selected from the group of AAVF7(SEQ ID NO: 8), AAVF12 (SEQ ID NO: 12), AAVF15 (SEQ ID NO: 16), AAVF17(SEQ ID NO: 13), variants, fragments, mutants and any combinationthereof. In certain embodiments, the one or more Clade F capsids orcapsid variants may comprise a polypeptide sequence having a percentsequence identity of at least 95% to a polypeptide sequence selectedfrom the group of AAVF5 (SEQ ID NO: 11), AAVF7 (SEQ ID NO: 8), AAVF12(SEQ ID NO: 12), AAVF15 (SEQ ID NO: 16), AAVF17 (SEQ ID NO: 13), AAVF9(SEQ ID NO: 10), AAVF16 (SEQ ID NO: 17), variants, fragments, mutantsand any combination thereof. In certain embodiments, the AAV Clade Fvector or AAV vector variant does not contain a promoter for the one ormore therapeutic nucleotide sequences. In certain embodiments, thetarget locus (target site) may be a safe harbor site. In certainembodiments, the safe harbor site may be the AAVS1 locus on chromosome19. In certain embodiments, the target locus (target site) may be alocus associated with a disease state as described herein. In certainembodiments, the cell may be a stem cell. In certain embodiments, thestem cell may be a hematopoietic stem cell, a pluripotent stem cell, anembryonic stem cell, or a mesenchymal stem cell.

According to certain embodiments, methods of treating a disease ordisorder in a subject by editing a genome of a cell of the subject areprovided. In certain embodiments, methods of treating a disease ordisorder in a subject by editing a genome of a cell of the subjectinclude the steps of transducing the cell of the subject with an AAVClade F vector or AAV vector variant as described herein andtransplanting the transduced cell into the subject, wherein thetransduced cell treats the disease or disorder. In certain embodiments,transduction of the cell may be performed without additional exogenousnucleases. In certain embodiments, AAV Clade F vectors or AAV vectorvariants may comprise one or more Clade F capsids or capsid variants(relative to an AAV9 capsid), an editing element (targeting cassette)comprising one or more therapeutic nucleotide sequences to be integratedinto a target locus (target site) of the genome, a 5′ homologous armpolynucleotide sequence flanking the editing element (targetingcassette) and having homology to a region that is upstream of the targetlocus (target site), and a 3′ homologous arm polynucleotide sequenceflanking the editing element (targeting cassette) and having homology toa region that is downstream of the target locus (target site). Theediting element (target cassette) may be contained within a correctiongenome as described herein comprising ITRs as described herein. Incertain embodiments, the Clade F capsids or capsid variants may comprisea polypeptide sequence selected from the group of AAVF7 (SEQ ID NO: 8),AAVF12 (SEQ ID NO: 12), AAVF15 (SEQ ID NO: 16), AAVF17 (SEQ ID NO: 13),variants, fragments, mutants and any combination thereof. In certainembodiments, the one or more Clade F capsids or capsid variants maycomprise a polypeptide sequence selected from the group of AAVF5 (SEQ IDNO: 11), AAVF7 (SEQ ID NO: 8), AAVF12 (SEQ ID NO: 12), AAVF15 (SEQ IDNO: 16), AAVF17 (SEQ ID NO: 13), AAVF9 (SEQ ID NO: 10), AAVF16 (SEQ IDNO: 17), variants, fragments, mutants and any combination thereof. Incertain embodiments, the one or more Clade F capsids or capsid variantsmay comprise a polypeptide sequence having a percent sequence identityof at least 95% to a polypeptide sequence selected from the group ofAAVF7 (SEQ ID NO: 8), AAVF12 (SEQ ID NO: 12), AAVF15 (SEQ ID NO: 16),AAVF17 (SEQ ID NO: 13), variants, fragments, mutants and any combinationthereof. In certain embodiments, the one or more Clade F capsids orcapsid variants may comprise a polypeptide sequence having a percentsequence identity of at least 95% to a polypeptide sequence selectedfrom the group of AAVF5 (SEQ ID NO: 11), AAVF7 (SEQ ID NO: 8), AAVF12(SEQ ID NO: 12), AAVF15 (SEQ ID NO: 16), AAVF17 (SEQ ID NO: 13), AAVF9(SEQ ID NO: 10), AAVF16 (SEQ ID NO: 17), variants, fragments, mutantsand any combination thereof. In certain embodiments, the AAV Clade Fvector or AAV vector variant does not contain a promoter for the one ormore therapeutic nucleotide sequences. In certain embodiments, thetarget locus (target site) may be a safe harbor site. In certainembodiments, the safe harbor site may be the AAVS1 locus on chromosome19. In certain embodiments, the target locus (target site) may be alocus associated with a disease state as described herein. In certainembodiments, the cell may be a stem cell. In certain embodiments, thestem cell may be a hematopoietic stem cell, a pluripotent stem cell, anembryonic stem cell, or a mesenchymal stem cell. In certain embodiments,the disease or disorder may be caused by one or more mutations in thecell genome. In certain embodiments, the disease or disorder may beselected from an inherited metabolic disease, lysosomal storage disease,mucopolysaccharidodosis, immunodeficiency disease, and hemoglobinopathydisease and infection.

Also disclosed herein are methods of treating a disease or disorder in asubject by in vivo genome editing by directly administering the AAVClade F vector or AAV vector variant as described herein to the subject.In certain embodiments, methods of treating a disease or disorder in asubject by in vivo genome editing of a cell of the subject by directlyadministering an AAV Clade F vector or AAV vector variant to the subjectare disclosed. In certain embodiments, the AAV Clade F vector or AAVvector variant may comprise one or more Clade F capsids or capsidvariants (relative to an AAV9 capsid), an editing element (targetingcassette) comprising one or more therapeutic nucleotide sequences to beintegrated into a target locus (target site) of the genome, a 5′homologous arm polynucleotide sequence flanking the editing element(targeting cassette) and having homology to a region that is upstream ofthe target locus (target site), and a 3′ homologous arm polynucleotidesequence flanking the editing element (targeting cassette) and havinghomology to a region that is downstream of the target locus (targetsite), wherein the vector transduces a cell of the subject andintegrates the one or more therapeutic nucleotide sequences into thegenome of the cell. The editing element (target cassette) may becontained within a correction genome as described herein comprising ITRsas described herein. In certain embodiments, the one or more Clade Fcapsids or capsid variants may comprise a polypeptide sequence selectedfrom the group of AAVF1 (SEQ ID NO: 2), AAVF2 (SEQ ID NO: 3), AAVF11(SEQ ID NO: 4), AAVF3 (SEQ ID NO: 5), AAVF4 (SEQ ID NO: 6), AAVF6 (SEQID NO: 7), AAVF7 (SEQ ID NO: 8), AAVF8 (SEQ ID NO: 9), AAVF9 (SEQ ID NO:10), AAVF5 (SEQ ID NO: 11), AAVF12 (SEQ ID NO: 12), AAVF17 (SEQ ID NO:13), AAVF13 (SEQ ID NO: 14), AAVF14 (SEQ ID NO: 15), AAVF15 (SEQ ID NO:16), AAVF16 (SEQ ID NO: 17), variants, fragments, mutants, and anycombination thereof. In certain embodiments, the AAV Clade F vector orAAV vector variant does not contain a promoter for the one or moretherapeutic nucleotide sequences. In certain embodiments, the targetlocus (target site) may be a safe harbor site. In certain embodiments,the safe harbor site may be the AAVS1 locus on chromosome 19. In certainembodiments, the target locus (target site) may be a locus associatedwith a disease state as described herein. In certain embodiments, thecell may be a stem cell. In certain embodiments, the stem cell may be ahematopoietic stem cell, a pluripotent stem cell, an embryonic stemcell, or a mesenchymal stem cell. In certain embodiments, the disease ordisorder may be caused by one or more mutations in the cell genome. Incertain embodiments, the disease or disorder may be selected from aninherited metabolic disease, lysosomal storage disease,mucopolysaccharidodosis, immunodeficiency disease, and hemoglobinopathydisease and infection. Also disclosed herein are kits comprising one ormore AAV Clade F vectors or AAV vector variants for editing the genomeof a cell.

BRIEF DESCRIPTION OF THE DRAWINGS

This application contains at least one drawing executed in color. Copiesof this application with color drawing(s) will be provided by the Officeupon request and payment of the necessary fees.

FIG. 1 shows the alignment of Clade F AAV capsid variant polypeptidesequences in comparison to AAV9. A corresponding alignment of Clade FAAV capsid variant polynucleotide sequences is provided in FIG. 1 of USPatent Publication Number US20130096182A1.

FIG. 2 is a chart listing some of the nucleotide mutations in the capsidof each sequence, including the base change, the amino acid change, andwhether it is in VP1 or VP3.

FIG. 3 shows a schematic of a portion of one set of donor ITR-AAVS1-FPvector constructs that were used for genome editing. The AAV vectorcontained 5′ homology and 3′ homology arms, and regulatory elements,which included a 2A sequence, splice acceptor sequence, andpolyadenylation sequence. Yellow fluorescent protein (“YFP” or “FP”) wasused as the transgene. AAV2 ITRs flanked the homologous arms and thevector genome was packaged in AAVF capsids to form the AAVF-AAVS1-FPdonor vectors. Importantly, the vector containing the FP gene does notcontain a promoter to drive expression. The FP gene will only beexpressed if it integrates correctly into AAVS1, downstream from anendogenous chromosomal promoter.

FIGS. 4A-4B show a schematic map of the targeted chromosomal AAVS1 locusand the edited AAVS1 locus that was the target site for transgeneintegration mediated by the AAVF vector. The top schematic (FIG. 4A),“Wild type AAVS1 locus”, illustrates the wild-type AAVS1 locus thatcontains a 5′ homology arm and a 3′ homology arm, but does not contain atransgene. Amplification with primers located outside of the homologyregion using an “OUT Forward Primer Region” primer and an “OUT ReversePrimer Region” primer results in a fragment ˜1.9 kb long (see linelabeled “Fragment 1”), which indicates that the fragment does notcontain an integrated transgene. The bottom schematic (FIG. 4B), “EditedAAVS1 locus”, illustrates the edited AAVS1 locus which contains a 5′homology arm, regulatory elements, an integrated transgene, and the 3′homology arm. Amplification with primers located outside of the homologyregion using an “OUT Forward Primer Region” primer and an “OUT ReversePrimer Region” primer results in a fragment ˜3.0 kb long (see linelabeled “Fragment 2”), which indicates that the fragment contains atransgene. Amplification of the 5′ junction region (the junction betweenthe 5′ homology arm and the transgene) using an “OUT Forward PrimerRegion” primer and an “In Reverse Primer” results in a fragment ˜1.7 kblong (see line labeled “Fragment 3”). Amplification of the 3′ junctionregion (the junction between the transgene and the 3′ homology arm)using an “OUT Reverse Primer Region” primer and an “In Forward Primer”results in a fragment ˜1.2 kb long (see line labeled “Fragment 4”). Ifthe transgene is not integrated, there is no resulting product uponamplification of the 5′ junction region or the 3′ junction region.

FIGS. 5A-5E show representative scatter plots from flow cytometricanalyses of YFP expression in K562 cells 24 hours after transduction.Cells were transduced with the AAVF7 FP vector at a variety ofmultiplicity of infections (MOIs) (A) Cells not transduced with anyvector (untransduced), (B) 50,000 MOI, (C) 100,000 MOI, (D) 200,000 MOI,and (E) 400,000 MOI. Data shown is from representative samples. Eventsabove the line of demarcation within each scatter plot represent FPexpressing cells, indicating that in these cells, the promoterless FPgene from the Donor ITR-AAVS1-FP vector integrated correctly into AAVS1in the human chromosome 19, downstream from the endogenous chromosomalpromoter.

FIGS. 6A-6E show representative scatter plots from flow cytometricanalyses of YFP expression in K562 cells 72 hours after transduction.Cells were transduced with the AAVF7 FP vector at variety ofmultiplicity of infections (MOIs) (A) Cells not transduced with anyvector (untransduced), (B) 50,000 MOI, (C) 100,000 MOI, (D) 200,000 MOI,and (E) 400,000 MOI. Events above the line of demarcation representcells with correctly targeted integration of the promoterless FP gene inthe Donor ITR-AAVS1-FP vector.

FIGS. 7A-7B show the average percentage of YFP expression followingtargeted integration of the promoterless YFP transgene in the AAVS1locus in CD34+K562 leukemic cells. (A) A bar graph showing YFPexpression 24 hours post-transduction with AAVF7 vector in cells withMOIs of 50,000; 100,000; 150,000; 200,000; 300,000; and 400,000. (B) Abar graph showing YFP expression 72 hours post-transduction with AAVF7vector in cells with MOIs of 50,000; 100,000; 150,000; 200,000; and400,000. Each bar represents data compiled from up to 7 samples.

FIGS. 8A-8B show PCR confirmation of targeted integration of the YFPtransgene into AAVS1 locus in K562 cells. A) Gel showing amplified DNAfrom representative samples from K562 cells with no template,untransduced, or transduced with AAVF7 FP vector at an MOI of 100,000.Lane 1: DNA ladder, lane 2: no template control, lane 3: untransducedcontrol, and lane 4: AAVF7 FP transduced K562. Arrows point to eitherthe FP integrated AAVS1 ˜3.1 kb fragment or the non-integrated AAVS1˜1.9 kb fragment. B) Gel showing amplified DNA from representativesamples from K562 cells with no template, untransduced, or transducedwith AAVF7 FP vector with an MOI of 100,000. Lane 1: DNA ladder, lane 2:no template control, lane 3: untransduced control, lane 4: AAVF7 FPvector transduced K562. The arrows point to either the amplified FPintegrated AAVS1 ˜3.1 kb fragment or the amplified non-integrated AAVS1˜1.9 kb fragment.

FIGS. 9A-9C show representative scatter plots of YFP expression inprimary CD34+ cells after targeted integration 1 day post-transductionwith AAVF FP vectors. (A) Cells not transduced with any vector(untransduced), (B) cells transduced with AAVF7 FP vector, and (C) cellstransduced with AAVF17 FP vector. Cells transduced with either AAVF7 orAAVF17 vector showed a significant amount of YFP expression comparedwith the untransduced cells (compare B and C, respectively, with A). YFPexpression in (B) and (C) indicates that the promoterless FP genedelivered by the AAVF vector accurately integrated into the chromosomalAAVS1 locus.

FIGS. 10A-10C show representative scatter plots of YFP expression inprimary CD34+ cells after targeted integration 4 days post-transductionwith AAVF FP vectors. (A) Cells not transduced with any vector(untransduced), (B) cells transduced with AAVF7 FP vector, and (C) cellstransduced with AAVF17 FP vector. Cells transduced with either AAVF7 orAAVF17 FP vector showed a significant amount of YFP expression comparedwith the untransduced cells (compare B and C, respectively, with A),indicating accurate targeted integration of the gene delivered by theAAVF vector.

FIGS. 11A-11C show representative scatter plots of YFP expression inprimary CD34+ cells after targeted integration 18 days post-transductionwith AAVF FP vectors from representative samples. (A) Cells nottransduced with any vector (untransduced), (B) cells transduced withAAVF7 FP vector, and (C) cells transduced with AAVF17 FP vector. Cellstransduced with either AAVF7 or AAVF17 FP vector showed a significantamount of YFP expression compared with the untransduced cells (compare Band C, respectively, with A).

FIGS. 12A-12B show YFP expression in primary CD34+ cells after targetedintegration. (A) A table showing the percentage of YFP positive cellsfor untransduced cells and cells transduced with either AAVF7 FP vectoror AAVF17 FP vector at 4, 18, 20, and 39 days post-transduction. (B) Aline graph showing the frequency of YFP expressing primary CD34+ cellsat 4, 18, 20, and 39 days post AAVF FP transduction with an MOI of100,000. The line with diamonds represents untransduced cells, the linewith squares represents cells transduced with AAVF7 FP vector, and theline with triangles represents cells transduced with AAVF17 FP vector.

FIG. 13 shows PCR confirmation of targeted integration into the AAVS1locus in primary CD34+ cells. A gel showing amplified DNA fromrepresentative samples of primary CD34+ cells with no template,untransduced, or transduced with AAVF7 FP vector with an MOI of 150,000.Lane 1: DNA ladder, lane 2: no template control, lane 3: untransducedcontrol, lane 4: DNA marker, and lane 5: AAVF7 FP vector transduced. Thearrow points to the FP integrated AAVS1 (˜1.7 kb fragment) showing theamplification product of the 5′ junction region. Inset to the left showsthe DNA ladder that was loaded in lane 1.

FIG. 14 shows sequence confirmation of targeted integration of YFP genesequences in the AAVS1 locus beginning at the OUT Forward Primer Region.Sequencing results indicate that the YFP gene was present and wasintegrated correctly into the AAVS1 locus.

FIG. 15 shows sequence confirmation of targeted integration of YFPsequences in the AAVS1 locus beginning near the 5′ homology arm.Sequencing results indicate that the YFP gene was present and wasintegrated into the AAVS1 locus.

FIG. 16 shows sequence confirmation of targeted integration of YFPsequences in the AAVS1 locus beginning near the 5′ region of theregulatory elements. Sequencing results indicate that the YFP gene waspresent and was integrated into the AAVS1 locus.

FIG. 17 shows sequence confirmation of targeted integration of YFPsequences in the AAVS1 locus beginning near the 3′ region of theregulatory elements. Sequencing results indicate that the YFP gene waspresent and was integrated into the AAVS1 locus.

FIG. 18 shows sequence confirmation of targeted integration of YFPsequences in the AAVS1 locus beginning near the 5′ region of thetransgene. Sequencing results indicate that the YFP gene was present andwas integrated into the AAVS1 locus.

FIG. 19 shows sequence confirmation of targeted integration of YFPsequences in the AAVS1 locus beginning near the “IN Reverse Primer”region. Sequencing results indicate that the YFP gene was present andwas integrated into the AAVS1 locus.

FIG. 20 shows a schematic of the steps performed in the experiments inExample 4. One million human cord blood CD34+ cells were obtained (seeStep 1) and injected into sublethally-irradiated immune deficientNOD/SCID adult mice (see Step 2). Two hours after injection with CD34+cells, the mice were injected with AAVF-Luciferase vector (i.e.,AAVF7-Luciferase vector or AAVF17-Luciferase vector). Two to seven dayslater the mice were injected with AAVF-Venus vectors (i.e., AAVF7-Venusvector or AAVF17-Venus vector) (see Step 3). Finally, in vivo luciferaseexpression was measured 4 weeks post injection and Venus expression wasquantitated 6 weeks post-injection (see Step 4).

FIGS. 21A-21B show in vivo specific luciferase expression inrepresentative recipients. FIG. 21A shows that adult immune-deficientmice previously xenografted with human cord blood CD34+ HSCs thatreceived intravenous injections of AAVF-Luciferase vector displayedspecific luciferase expression in vertebrae, spleen, hips, and longbones, all sites of hematopoiesis after transplantation. Arrows indicateluciferase expression in vertebrae, spleen, liver, hips, and long bones.Flux for the liver and spleen was 4.08e9 and flux for the tail was1.74e9. FIG. 21B shows that adult immune-deficient mice that were notpreviously xenografted with human cord blood CD34+ HSCs that receivedintravenous injections of AAVF-Luciferase vector did not display highlevels of specific luciferase expression. Flux for the liver and spleenwas 1.47e8 and flux for the tail was 2.22e8.

FIGS. 22A-H show histograms illustrating flow cytometry data ofVenus-expressing human CD34+ or CD45+ cells in adult immune-deficientmice previously xenografted with human cord blood CD34+ HSCs thatreceived intravenous injections of either AAVF7-Venus or AAVF17-Venusvectors. FIG. 22A shows flow cytometry data from femoral CD34+ cells ofxenografted mice injected with AAVF7-Venus vector. 9.23% of engraftedhuman hematopoietic cells expressed Venus. FIG. 22B shows flow cytometrydata from femoral CD45+ cells of xenografted mice injected withAAVF7-Venus vector. 8.35% of engrafted human hematopoietic cellsexpressed Venus. FIG. 22C shows flow cytometry data from femoral CD34+cells of xenografted mice injected with AAVF17-Venus vector. 8.92% ofengrafted human hematopoietic cells expressed Venus. FIG. 22D shows flowcytometry data from femoral CD45+ cells of xenografted mice injectedwith AAVF17-Venus vector. 8.59% of engrafted human hematopoietic cellsexpressed Venus. FIG. 22E shows flow cytometry data from vertebral CD45+cells of xenografted mice injected with AAVF7-Venus vector. 15.3% ofengrafted human hematopoietic cells expressed Venus. FIG. 22F shows flowcytometry data from vertebral CD45+ cells of xenografted mice injectedwith AAVF17-Venus vector. 70.2% of engrafted human hematopoietic cellsexpressed Venus. FIG. 22G shows flow cytometry data from spleen CD45+cells of xenografted mice injected with AAVF7-Venus vector. 10.3% ofengrafted human hematopoietic cells expressed Venus. FIG. 22H shows flowcytometry data from spleen CD45+ cells of xenografted mice injected withAAVF17-Venus vector. 9.90% of engrafted human hematopoietic cellsexpressed Venus. Results from FIG. 22 are also provided in Table 5.

FIG. 23 shows a phylogram of the relationship of AAV Clade F virusesrelative to each other and other AAV strains. The phylogram is based onnucleotide sequence homology of the capsid genes of AAVF viruses (Smithet al, Mol Ther. 2014 September; 22(9):1625-34).

FIG. 24 shows a map of a single stranded AAV vector genome for theinsertion of a large DNA insert. The single stranded AAV2 genomecontained AAV2 ITRs, homology arms, regulatory sequences and thepromoterless Venus open reading frame (ORF). Venus is a fluorescentreporter protein. The promoterless Venus containing the Venus ORF isdownstream from a splice acceptor and 2A sequence. The Venus ORF isfollowed by a polyadenylation signal. Each homology arms is 800 bp longand targets Intron 1 of PPP1R12C gene on Chromosome 19

FIG. 25 shows a schematic of an insertion site of an editing moiety inAAVS1. The transgene cassette consisting of the Venus open reading frameand a splice acceptor site followed by 2A sequence is flanked on eitherside by homology arms. Homology arms are complementary to Intron 1 ofthe human PPP1R12C gene within the AAVS1 locus on chromosome 19 andmediate insertion of Venus into the site between the two homology arms.

FIGS. 26A-F show targeted genomic insertion of a large protein codingsequence by recombinant AAVF vectors in human cell lines and primarycells demonstrating that AAVF-mediated genome editing is robust and thatthere is efficient editing in Human CD34+ cells and cell lines. FIG.26A: CD34+ represents primary human CD34+ cytokine-primed peripheralblood stem cells. FIG. 26B: K562 is a human CD34+ erythroleukemia cellline. FIG. 26C: HepG2 is a human liver cell line. The percent of cellsdisplaying Venus expression, indicative of precise insertion, is shownfor FIGS. 26A-C. FIG. 26D shows representative flow profiles showing adistinct Venus expressing population of CD34+ cells after transductionwith recombinant AAVF viruses, as compared with untransduced cells. FIG.26E shows editing activity of AAVF7, AAVF12, AAVF15, AAVF17 and AAV9 ascompared with AAV6 and AAV8 in a K562 erythroleukemia line. FIG. 26Fshows editing activity of the same virus in HepG2, a liver cell line.Data shows the percent of cells displaying editing and Venus expression.

FIG. 27 shows a targeted integration assay for the detection of largeand small inserts into the AAVS1 locus on the human chromosome 19. Theschematic maps show the location of primers. The 5′ primer iscomplementary to chromosomal sequences. The 3′ primer is specific forthe insert. The specific amplicon is predicted to be 1.7 kb for thelarge insert and 1 kb for the small insert. The split primer pair(chromosomal and insert specific) lends specificity to the targetedintegration assay.

FIGS. 28A-E show that AAVF vectors mediate nucleotide substitution atspecified genomic sites. FIG. 28A shows maps of single stranded AAVvector genomes for the insertion of a 10 bp insert in intron 1 of thehuman PPPTR12C gene. This vector encodes a wild type left homology arm(HA-L) which contains an Nhe1 restriction enzyme recognition site(GCTAGC). The NS mut vector, was designed to change the TA sequence inthe left homology arm on chromosome 19 to AT. This change results in theconversion of an Nhe1 site to an Sph1 site, changing the sequence fromGCTAGC to GCATGC. FIG. 28B shows that the left homology arm wasamplified using a forward primer located in upstream chromosomalsequences and a reverse primer located in the 10 bp insert in Intron 1of the PPPTR12C gene on chromosome 19. The upper schematic designatesthe relative sizes of the expected fragments created when genomic DNAfrom K562 cells is edited using either the wild type or the NS Mut AAVFvectors. FIG. 28C is a gel that shows the actual amplicons derived fromgenomic DNA of K562 cells edited with a wild type AAVF vector. Lanesshow the uncut amplicon (Un), the amplicon cut with Nhe 1 (Nhe1) andwith Sph 1 (Sph 1). FIG. 28D shows gel electrophoresis of K562 DNA afterediting with AAVF7 or an AAVF17 vectors encoding either wild type or NSMut genomes. FIG. 28E shows gel electrophoresis of a hepatocellularcarcinoma cell line, HepG2 after editing with AAVF7 or an AAVF17 vectorsencoding either wild type or NS Mut genomes.

FIG. 29 shows a sequence analysis of DNA from cells edited with AAVF7and AAVF17 Wild type or NS Mut vectors.

FIG. 30 is a table that showing AAVF vectors mediate editing in bothdividing and non-dividing cells and that AAVF-mediated gene editing doesnot require DNA synthesis. The figure shows frequency of edited cellsexpressing Venus in the dividing and non-dividing subsets of primaryhuman CD34+ cells. The percentage of all CD34+ cells that were Venuspositive and either BrdU positive or negative was determined by flowcytometry. BrdU positive cells represent dividing cells and BrdUnegative cells represent non-dividing cells.

FIGS. 31A-C show efficient editing of engrafted human hematopoietic stemcells in vivo by systemically delivered AAVF vectors. FIG. 31A shows adiagram of the experimental design. Immune deficient NOD/SCID mice wereengrafted with human cord blood CD34+ hematopoietic stem cells. Cellswere allowed to engraft for 7 weeks prior to intravenous injection ofAAVF17-Venus. Hematopoietic cells were harvested from the vertebral andfemoral marrow and spleen of xenografted mice 12.5 weeks after AAVFinjection. Cells were analyzed by multi-color flow cytometry for Venusexpression as well as the presence of human-specific surface markers.Specifically, Venus expression was analyzed in the primitive CD34+ humanhematopoietic stem/progenitor cells, CD45+ human differentiatedmononuclear hematopoietic cells and glycophorin A+ cells of theerythroid lineage. FIG. 31B shows a schematic of the differentiationpathway of the human erythroid lineage, from CD34+ progenitor cells tothe glycophorin A+ red blood cells. FIG. 31C shows flow cytometricprofiles of long term engrafted human cells in the marrow and spleencells of xenografted mice, 20 weeks after transplantation. Cells wereanalyzed for both expression of Venus, a marker of editing as well asspecific human cell surface markers.

FIGS. 32A and B are a summary of in vivo data following intravenousinjection of AAVF vectors into immune deficient mice xenografted withhuman cord blood CD34+ hematopoietic stem/progenitor cells. Venusexpression reflects targeted insertion of the promoterless Venuscassette into Intron 1 of the human PPP1R12C gene in in vivo engraftedhuman hematopoietic stem cells and their progeny. FIG. 32B is a summaryof the data in FIG. 32A. FIGS. 32A and B show that editing is long term,that editing is stably inherited (the insert is efficiently expressed indifferentiated progeny cells), that in vivo editing may be much moreefficient than ex vivo transduction followed by transplant, and thatprogeny of edited CD34+ cells retain Venus expression long term.

FIG. 33 shows a sequence analysis of targeted chromosomal insertion of apromoterless SA/2A venus ORF in K562 erythroleukemia cell line, primaryhuman cytokine-primed peripheral blood CD34+ cells (PBSC) and HepG2human liver cell line. Site-specifically integrated sequences wereamplified using a chromosome-specific primer and an insert-specificprimer. The amplified product was cloned into a TOPO-TA vector andsequenced using Sanger sequencing.

FIG. 34 shows a sequence analysis of targeted chromosomal insertion of a10 bp insert in primary human cytokine-primed peripheral blood CD34+cells and the HepG2 human liver cell line. Site-specifically integratedsequences were amplified using a chromosome-specific primer and aninsert-specific primer. The amplified product was cloned into a TOPO-TAvector and sequenced using Sanger sequencing.

FIG. 35 shows that AAVF targets integration of small inserts intoAAVS1-CD34+ cells.

FIG. 36 shows AAV targeting of promoterless Venus & RFLP in a HepG2(Hepatoma) cell line.

FIG. 37 shows representative femoral bone marrow flow cytometry graphsshowing total population sorted, backgating of CD34 and glycoA positivecells onto Venus positive and Venus negative populations, CD34/GlycoApositive cells in Venus negative populations, and glycoA positive andCD34 positive populations.

FIG. 38 shows representative spleen flow cytometry graphs showing totalpopulation sorted, backgating of CD34 and glycoA positive cells ontoVenus positive and Venus negative populations, CD34/GlycoA positivecells in Venus negative populations, and glycoA positive and CD34positive populations.

FIG. 39 shows maps of CBA-mCherry and AAS1-Venus vector genomes used todetermine the relative transduction versus editing efficiencies of AAVvectors.

FIGS. 40A and 40B show flow cytometric profiles of mCherry (FIG. 40A)and Venus (FIG. 40B) expression in human CD34+ cord blood cells. FIG.40C shows quantitation of mCherry and Venus expression in CD34+ cells 48hours after transduction. FIG. 40D shows a comparison of the relativeexpression of Venus to mCherry (Editing Ratio). Bars denote the ratio ofthe proportion of cells expressing Venus as a ratio of those expressingmCherry with the corresponding capsid. The black horizontal bar denotesa ratio of 1, which would indicate equal efficiencies of Venus:mCherryexpression.

DETAILED DESCRIPTION

Certain embodiments of the invention are described in detail, usingspecific examples, sequences, and drawings. The enumerated embodimentsare not intended to limit the invention to those embodiments, as theinvention is intended to cover all alternatives, modifications, andequivalents, which may be included within the scope of the presentinvention as defined by the claims. One skilled in the art willrecognize many methods and materials similar or equivalent to thosedescribed herein, which could be used in the practice of the presentinvention. Unless defined otherwise, all technical and scientific termsused herein have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs. Allpublications and/or patents are incorporated by reference as thoughfully set forth herein.

Provided herein are adeno-associated virus (AAV) Clade F vectors (e.g.,replication-defective AAVs comprising correction genomes enclosed in aClade F capsid) or AAV vector variants (e.g., replication-defective AAVscomprising capsid variants relative to AAV9 capsids) and related methodsthereof that were developed for precise editing of the genome of a cellusing homologous recombination without the need for addition ofexogenous nucleases. In certain embodiments, genome editing may include,without limitation, introducing insertions, deletions, alterations,point mutations or any combination thereof into the genome sequence of acell (e.g., a target locus of a mammalian chromosome). In certainembodiments, the AAV Clade F vectors or AAV vector variants and relatedmethods thereof provided herein may be used to insert one or morenucleotide sequences into a specific location of a cell genome withoutthe need for addition of exogenous nucleases prior to integration of theone or more nucleotide sequences. In certain embodiments, the Clade Fvectors or AAV vector variants and related methods thereof providedherein may be used to insert an internucleotide bond into a specificlocation of a cell genome without the need for addition of exogenousnucleases prior to integration of the internucleotide bond. Alsoprovided in certain embodiments are methods of treating a disease ordisorder in a subject by ex-vivo editing the genome of a cell of thesubject via transducing the cell with a Clade F vector or AAV vectorvariant as described herein and further transplanting the transducedcell into the subject to treat the disease or disorder of the subject.Also provided herein are methods of treating a disease or disorder in asubject by in vivo genome editing by directly administering the Clade Fvector or AAV vector variant as described herein to the subject. Alsoprovided herein are kits for genome editing of a cell comprising one ormore of the Clade F vectors or AAV vector variants described herein.

Homologous recombination using various AAV vectors (e.g., AAV2, AAV6,and AAV8) has been previously reported; however, the reportedefficiencies were very low—approximately 1 in a million cells. As shownin Examples 1 and 2 below, AAV Clade F vectors (or AAV vector variants)were used to reproducibly target gene insertion to specified chromosomallocations at significantly greater frequencies than previously seen. Forexample, targeted genome editing was achieved by transducing primarycells with AAV Clade F vectors (or AAV vector variants) resulting in theinsertion of the transgene into the genome of the primary cells atsurprisingly high frequencies, with approximately 10% of the primarycells displaying insertion of the transgene six weeks post-transduction.This frequency is 1,000 to 100,000 fold more efficient than previouslyreported (see, e.g., Khan, 2011). As shown in Examples 1 and 2 below,high level genome editing was achieved using primary human CD34+hematopoietic stem cells (K562) and CD34+ primary peripheralblood-derived human hematopoietic stem cells (PBSCs). Targeted geneinsertion was observed in both short term (one day) and long term (up toalmost six weeks) CD34+ cultures and was verified by transgeneexpression and sequence analysis. Furthermore, the Clade F vector or AAVvector variant targeted recombination as described herein allows forspecific genome engineering with no associated toxicity. As shown inExample 3 below, intravenous injection of AAV vectors pseudotyped withAAVF7 or AAVF17 resulted in transduction of human CD34+ hematopoieticstem and progenitor cells in vivo. As shown in Example 4 below, genomeediting was achieved both in cell culture and in vivo for both small(˜10 bps) and large inserts (˜800 bps) and was shown by sequencing to beprecisely integrated into the target locus. As shown in Example 5 below,genome editing of various human cell lines (e.g., fibroblasts,hepatocellular carcinoma cells, breast cancer cells, retinoblastomacells, leukemia cells and B cells) was achieved, demonstrating thatClade F vectors could be used to edit genomes in several distinct celltypes, such as fibroblasts, liver cells, breast cells, retinal cells,and B cells. As such, this technique has tremendous potential fortargeted genome editing in cells ex vivo as well as in vivo in specificorgans.

Provided herein are Clade F vectors (e.g., replication-defective AAVscomprising correction genomes enclosed in a Clade F capsid) or AAVvector variants (e.g., replication-defective AAVs comprising capsidvariants relative to AAV9 capsids) for editing a genome of a cell andmethods thereof (via recombination, and preferably without the use ofexogenous nucleases). In certain embodiments, genome editing mayinclude, without limitation, correction or insertion of one or moremutations in the genome, deletion of one or more nucleotides in thegenome, alteration of genomic sequences including regulatory sequences,insertion of one or more nucleotides including transgenes at safe harborsites or other specific locations in the genome, or any combinationthereof. In certain embodiments, genome editing using the Clade Fvectors or AAV vector variants and methods thereof as described hereinmay result in the induction of precise alterations of one or moregenomic sequences without inserting exogenous viral sequences or otherfootprints.

In some aspects, the disclosure provides a replication-defectiveadeno-associated virus (AAV) comprising a correction genome enclosed ina capsid as described herein, e.g., an AAV Clade F capsid. In someembodiments, a “correction genome” is a nucleic acid molecule thatcontains an editing element as described herein along with additionalelement(s) (e.g., a 5′ inverted terminal repeat (5′ ITR) nucleotidesequence, or a fragment thereof, and a 3′ inverted terminal repeat (3′ITR) nucleotide sequence, or a fragment thereof) sufficient forencapsidation within a capsid as described herein. It is to beunderstood that the term “correction genome” does not necessarilyrequire that an editing element contained within the correction genomewill “correct” a target locus in a genome, once integrated into thetarget locus (e.g., correction of target locus containing a mutation byreplacement with a wild-type sequence). Accordingly, in someembodiments, a correction genome may contain an editing element whichmay comprise a nucleotide sequence that is additive to the target locus(e.g., the target locus is the 3′ end of a first open reading frame andthe editing element is a second open reading frame that, when integratedinto the target locus, will create a gene that encodes a fusionprotein).

In some embodiments, the replication-defective adeno-associated virus(AAV) comprises a correction genome, the correction genome comprising(a) an editing element selected from an internucleotide bond or anucleotide sequence for integration into a target locus of a mammalianchromosome, (b) a 5′ homologous arm nucleotide sequence 5′ of theediting element, having homology to a 5′ region of the mammalianchromosome relative to the target locus, and (c) a 3′ homologous armnucleotide sequence 3′ of the editing element, having homology to a 3′region of the mammalian chromosome relative to the target locus. In someembodiments, the replication-defective AAV comprises a correctiongenome, the correction genome comprising an editing element nucleotidesequence for integration into a target locus of a mammalian chromosome,the correction genome having an essential absence of a promoteroperatively linked to the editing element nucleotide sequence. In someembodiments, the replication-defective AAV comprises a correctiongenome, the correction genome comprising an editing element selectedfrom an internucleotide bond or a nucleotide sequence for integrationinto a target locus of a mammalian chromosome in a cell; the AAV havinga chromosomal integration efficiency of at least about 1% (e.g., atleast about 2%, at least about 5%, at least about 10%, at least about20%, at least about 30%, at least about 40%, at least about 50%, atleast about 60%, at least about 70%, at least about 80%, or at leastabout 90%) for integrating the editing element into the target locus ofthe mammalian chromosome in the cell. In some embodiments, thereplication-defective AAV comprises a correction genome, the correctiongenome comprising an editing element selected from an internucleotidebond or a nucleotide sequence for integration into a target locus of amammalian chromosome in a cell; the AAV having a chromosomal integrationefficiency of at least about 1% (e.g., at least about 2%, at least about5%, at least about 10%, at least about 20%, at least about 30%, at leastabout 40%, at least about 50%, at least about 60%, at least about 70%,at least about 80%, or at least about 90%) in the absence of anexogenous nuclease for integrating the editing element into the targetlocus of the mammalian chromosome in the cell. In some embodiments ofany one of the correction genomes, the correction genome has anessential absence of a promoter operatively linked to the editingelement nucleotide sequence. In some embodiments of any one of thecorrection genomes, the correction genome further comprises an exogenouspromoter operatively linked to the editing element. In some embodimentsof any one of the replication-defective AAVs, the AAV has a chromosomalintegration efficiency of at least about 1%, at least about 2%, at leastabout 3%, at least about 4%, at least about 5%, at least about 10%, atleast about 20%, at least about 30%, at least about 40%, at least about50%, at least about 60%, at least about 70%, at least about 80%, or atleast about 90% for integrating an editing element into a target locusof a mammalian chromosome in a cell.

Other aspects of the disclosure relate to a gene editing vectorcomprising a replication-defective adeno-associated virus (AAV)comprising a correction genome enclosed in an AAV capsid, the correctiongenome as described herein (e.g., comprising an editing element selectedfrom an internucleotide bond or a nucleotide sequence for integrationinto a target locus of a mammalian cell chromosome; a 5′ homologous armnucleotide sequence 5′ of the editing element having homology to a 5′region of the chromosome relative to the target locus; a 3′ homologousarm nucleotide sequence 3′ of the editing element having homology to a3′ region of the chromosome relative to the target locus); wherein theAAV has a chromosomal integration efficiency of at least 10% (e.g., atleast 15%, at least 20%, at least 30%, at least 40%, at least 50%, atleast 60%, at least 70%, at least 80%, or at least 90%) for integratingan editing element as described herein into a target locus as describedherein. In some embodiments, the chromosomal integration efficiency isat least 10% (e.g., at least 15%, at least 20%, at least 30%, at least40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least90%) for integrating an editing element as described herein into atarget locus as described herein in the absence of an exogenousnuclease.

A correction genome as described herein can comprise a 5′ invertedterminal repeat (5′ ITR) nucleotide sequence 5′ of the 5′ homologous armnucleotide sequence, and a 3′ inverted terminal repeat (3′ ITR)nucleotide sequence 3′ of the 3′ homologous arm nucleotide sequence. Insome embodiments, the 5′ ITR nucleotide sequence and the 3′ ITRnucleotide sequence are substantially identical (e.g., at least 90%, atleast 95%, at least 98%, at least 99% identical or 100% identical) to anAAV2 virus 5′ITR and an AAV2 virus 3′ ITR, respectively. In someembodiments, the 5′ ITR nucleotide sequence has at least 95% (e.g., atleast 96%, at least 97%, at least 98%, at least 99%, or 100%) sequenceidentity to SEQ ID NO:36, and the 3′ ITR nucleotide sequence has atleast 95% (e.g., at least 96%, at least 97%, at least 98%, at least 99%,or 100%) sequence identity to SEQ ID NO:37. In some embodiments, the 5′ITR nucleotide sequence and the 3′ ITR nucleotide sequence aresubstantially identical (e.g., at least 90%, at least 95%, at least 98%,at least 99% identical or 100% identical) to an AAV5 virus 5′ITR and anAAV5 virus 3′ ITR, respectively. In some embodiments, the 5′ ITRnucleotide sequence has at least 95% (e.g., at least 96%, at least 97%,at least 98%, at least 99%, or 100%) sequence identity to SEQ ID NO:38,and the 3′ ITR nucleotide sequence has at least 95% (e.g., at least 96%,at least 97%, at least 98%, at least 99%, or 100%) sequence identity toSEQ ID NO:39. In some embodiments, the 5′ ITR nucleotide sequence andthe 3′ ITR nucleotide sequence are substantially mirror images of eachother (e.g., are mirror images of each other except for at 1, 2, 3, 4 or5 nucleotide positions in the 5′ or 3′ ITR).

Exemplary AAV2 5′ ITR- (SEQ ID NO: 36)ttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgcagagagggagtggccaactccatcactaggggttcct Exemplary AAV2 3′ ITR-(SEQ ID NO: 37) aggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgcagagagggagtggccaa Exemplary AAV5 5′ ITR-(SEQ ID NO: 38) ctctcccccctgtcgcgttcgctcgctcgctggctcgtttgggggggtggcagctcaaagagctgccagacgacggccctctggccgtcgcccccccaaacgagccagcgagcgagcgaacgcgacaggggggagagtgccacactctca agcaagggggttttgtaExemplary AAV5 3′ ITR- (SEQ ID NO: 39)tacaaaacctccttgcttgagagtgtggcactctcccccctgtcgcgttcgctcgctcgctggctcgtttgggggggtggcagctcaaagagctgccagacgacggccctctggccgtcgcccccccaaacgagccagcgagcgagcgaa cgcgacaggggggagag

In some embodiments, a correction genome as described herein is no morethan 7 kb (kilobases), no more than 6 kb, no more than 5 kb, or no morethan 4 kb in size. In some embodiments, a correction genome as describedherein is between 4 kb and 7 kb, 4 kb and 6 kb, 4 kb and 5 kb, or 4.1 kband 4.9 kb.

In certain embodiments, AAV Clade F vectors or AAV vector variants forediting a genome of a cell comprise one or more Clade F capsids orcapsid variants (variant relative to an AAV9 capsid). In certainembodiments, AAV Clade F vectors or AAV vector variants for editing agenome of a cell comprise one or more AAV Clade F capsids. In certainembodiments, a donor vector may be packaged into the Clade F capsids orcapsid variants described herein according to a standard AAV packagingmethod resulting in formation of the AAV Clade F vector or AAV vectorvariant (see e.g., Chatterjee, 1992). In certain embodiments, the one ormore Clade F capsids or capsid variants influence the tropism of the AAVClade F vector or AAV vector variant for a particular cell.

According to certain embodiments, the one or more Clade F capsids orcapsid variants may be derived from human stem cell-derived AAV. It hasbeen previously shown that that cytokine-primed peripheral blood CD34+stem cells from healthy donors harbor endogenous natural AAV sequencesin their genome (see, e.g., US Patent Publication Number US20130096182A1and US20110294218A1). The efficacy of the AAV isolate variants (variantrelative to AAV9) has been previously demonstrated, including theefficacy of individual capsid nucleotides and proteins for use in celltransduction (see, e.g., US Patent Publication Number US20130096182A1and US20110294218A1).

Full length AAV capsid variant genes (variant relative to AAV9) from thedonors harboring endogenous natural AAV sequences in their genome wereisolated and sequenced. The polynucleotide and polypeptide sequences ofthe capsid variants are provided in FIG. 1 and in U.S. patentapplication Ser. No. 13/668,120, filed Nov. 2, 2012, published as USPatent Publication Number US20130096182A1, and U.S. patent applicationSer. No. 13/097,046, filed Apr. 28, 2011, US20110294218A1, published asUS Patent Publication Number US20130096182A1, which issued on Jan. 14,2014 as U.S. Pat. No. 8,628,966, all of which are hereby incorporated byreference in their entirety, as if fully set forth herein. In certainembodiments, the AAV Clade F vectors or AAV vector variants describedherein may comprise one or more Clade F capsids or capsid variantscomprising a polynucleotide sequence selected from the group of AAVF1(SEQ ID NO: 20), AAVF2 (SEQ ID NO: 21), AAVF3 (SEQ ID NO: 22), AAVF4(SEQ ID NO: 23), AAVF5 (SEQ ID NO: 25), AAVF11 (SEQ ID NO: 26), AAVF7(SEQ ID NO: 27), AAVF8 (SEQ ID NO: 28), AAVF9 (SEQ ID NO: 29), AAVF12(SEQ ID NO: 30), AAVF13 (SEQ ID NO: 31), AAVF14 (SEQ ID NO: 32), AAVF15(SEQ ID NO: 33), AAVF16 (SEQ ID NO: 34), AAVF17 (SEQ ID NO: 35),variants, fragments, mutants, and any combination thereof. In certainembodiments, the AAV Clade F vectors or AAV vector variants describedherein may comprise one or more Clade F capsids or capsid variantscomprising a polypeptide sequence selected from the group of AAVF1 (SEQID NO: 2), AAVF2 (SEQ ID NO: 3), AAVF11 (SEQ ID NO: 4), AAVF3 (SEQ IDNO: 5), AAVF4 (SEQ ID NO: 6), AAVF6 (SEQ ID NO: 7), AAVF7 (SEQ ID NO:8), AAVF8 (SEQ ID NO: 9), AAVF9 (SEQ ID NO: 10), AAVF5 (SEQ ID NO: 11),AAVF12 (SEQ ID NO: 12), AAVF17 (SEQ ID NO: 13), AAVF13 (SEQ ID NO: 14),AAVF14 (SEQ ID NO: 15), AAVF15 (SEQ ID NO: 16), AAVF16 (SEQ ID NO: 17),variants, fragments, mutants, and any combination thereof (see, e.g.,FIG. 1 ).

According to certain embodiments, the polynucleotide or polypeptidesequences of the Clade F capsids or capsid variants may have at leastabout 95%, 96%, 97%, more preferably about 98%, and most preferablyabout 99% sequence identity to the sequences taught in the presentspecification. Percentage identity may be calculated using any of anumber of sequence comparison programs or methods such as the Pearson &Lipman, Proc. Natl. Acad. Sci. USA, 85:2444 (1988), and programsimplementing comparison algorithms such as GAP, BESTFIT, FASTA, orTFASTA (from the Wisconsin Genetics Software Package, Genetics ComputerGroup, 575 Science Drive, Madison, Wis.), or BLAST, available throughthe National Center for Biotechnology Information web site.

The Clade F capsids or capsid variant sequences may be modified at oneor more positions in the V1 and/or V3 cap genes, these genes orfunctional portions of the genes can be used separately or together inany of the AAV Clade F vectors or AAV vector variants and methodsdescribed herein. Cap genes, V1, V2, and V3, may be substituted out frommultiple mutated sequences, and are typically used in a colinear fashionV1-V2-V3. However the sequences may be truncated such as partialV1-V2-V3 or V1-V3 or V1-V1-V2-V3. For example, one sequence could be V1of (AAVF8)-V2 of (AAVF4)-V3 of AAVF14. Preferably, the Clade F capsidsor capsid variants transduce the target cells on a level at or higherthan AAV2.

In certain embodiments, the one or more capsid variants may comprise acombination of one or more V1, V2, and V3 polynucleotide sequences ofcapsid variants (e.g., SEQ ID NOs: 20-35, variant relative to AAV9capsid), the AAV9 capsid (SEQ ID NO: 18), the AAV2 capsid (SEQ ID NO:19), variants, fragments, or mutants thereof. In certain embodiments,the one or more or capsid variants may comprise a combination of one ormore V1, V2, and V3 polynucleotide sequences of capsid variants (SEQ IDNOs: 20-35, variant relative to AAV9 capsid), any other known AAVcapsids, variants, fragments, or mutants thereof.

In certain embodiments, the one or more Clade F capsids or capsidvariants may comprise a combination of V1, V2, and V3 polypeptidesequences of the capsid variants (SEQ ID NOs: 2-17, variant relative toAAV9 capsid), the AAV9 capsid (SEQ ID NO: 1), variants, fragments, ormutants thereof. In certain embodiments, the one or more capsid variantsmay comprise a combination of V1, V2, and V3 polypeptide sequences ofthe Clade F capsid variants (SEQ ID NOs: 2-17, variant relative toAAV9), any other known AAV capsid, variants, fragments, or mutantsthereof.

In some embodiments, an AAV Clade F vector or AAV vector variant forediting a genome of a cell comprises a AAV Clade F capsid. In someembodiments, an “AAV Clade F capsid” refers to a capsid that has an AAVVP1, VP2, and/or VP3 sequence that has at least 86% (e.g., at least 86%,at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99%) sequence identity with the AAVVP1, VP2, and/or VP3 sequence of AAV9, respectively. Exemplary Clade Fcapsids include AAVF1-17 (also referred to herein as AAVHSC1-17), AAV9,AAVHU31, AAVHU32, and AAVAnc110 (see, e.g., Zinn et al. In SilicoReconstruction of the Viral Evolutionary Lineage Yields a Potent GeneTherapy Vector (2015) Cell Reports, Vol 12, pp. 1056-1068).

In some embodiments, an AAV Clade F capsid comprises at least one or atleast two proteins selected from Clade F VP1, Clade F VP2 and Clade FVP3. In some embodiments, an AAV Clade F capsid comprises Clade F VP1,Clade F VP2 and Clade F VP3 proteins.

Exemplary AAV VP1, VP2, and VP3 protein sequences of AAV Clade F capsidsare provided in the below table.

AAV capsid VP1 VP2 VP3 AAV9 Amino acids 1 to 736 Amino acids 138 toAmino acids 203 to of SEQ ID NO: 1 736 of SEQ ID NO: 1 736 of SEQ ID NO:1 AAVF1 Amino acids 1 to 736 Amino acids 138 to Amino acids 203 to ofSEQ ID NO: 2 736 of SEQ ID NO: 2 736 of SEQ ID NO: 2 AAVF2 Amino acids 1to 736 Amino acids 138 to Amino acids 203 to of SEQ ID NO: 3 736 of SEQID NO: 3 736 of SEQ ID NO: 3 AAVF3 Amino acids 1 to 736 Amino acids 138to Amino acids 203 to of SEQ ID NO: 5 736 of SEQ ID NO: 5 736 of SEQ IDNO: 5 AAVF4 Amino acids 1 to 736 Amino acids 138 to Amino acids 203 toof SEQ ID NO: 6 736 of SEQ ID NO: 6 736 of SEQ ID NO: 6 AAVF5 Aminoacids 1 to 736 Amino acids 138 to Amino acids 203 to of SEQ ID NO: 11736 of SEQ ID NO: 11 736 of SEQ ID NO: 11 AAVF6 Amino acids 1 to 736Amino acids 138 to Amino acids 203 to of SEQ ID NO: 7 736 of SEQ ID NO:7 736 of SEQ ID NO: 7 AAVF7 Amino acids 1 to 736 Amino acids 138 toAmino acids 203 to of SEQ ID NO: 8 736 of SEQ ID NO: 8 736 of SEQ ID NO:8 AAVF8 Amino acids 1 to 736 Amino acids 138 to Amino acids 203 to ofSEQ ID NO: 9 736 of SEQ ID NO: 9 736 of SEQ ID NO: 9 AAVF9 Amino acids 1to 736 Amino acids 138 to Amino acids 203 to of SEQ ID NO: 10 736 of SEQID NO: 10 736 of SEQ ID NO: 10 AAVF10 Amino acids 1 to 736 Amino acids138 to Amino acids 203 to of SEQ ID NO: 3 736 of SEQ ID NO: 3 736 of SEQID NO: 3 AAVF11 Amino acids 1 to 736 Amino acids 138 to Amino acids 203to of SEQ ID NO: 4 736 of SEQ ID NO: 4 736 of SEQ ID NO: 4 AAVF12 Aminoacids 1 to 736 Amino acids 138 to Amino acids 203 to of SEQ ID NO: 12736 of SEQ ID NO: 12 736 of SEQ ID NO: 12 AAVF13 Amino acids 1 to 736Amino acids 138 to Amino acids 203 to of SEQ ID NO: 14 736 of SEQ ID NO:14 736 of SEQ ID NO: 14 AAVF14 Amino acids 1 to 736 Amino acids 138 toAmino acids 203 to of SEQ ID NO: 15 736 of SEQ ID NO: 15 736 of SEQ IDNO: 15 AAVF15 Amino acids 1 to 736 Amino acids 138 to Amino acids 203 toof SEQ ID NO: 16 736 of SEQ ID NO: 16 736 of SEQ ID NO: 16 AAVF16 Aminoacids 1 to 736 Amino acids 138 to Amino acids 203 to of SEQ ID NO: 17736 of SEQ ID NO: 17 736 of SEQ ID NO: 17 AAVF17 Amino acids 1 to 736Amino acids 138 to Amino acids 203 to of SEQ ID NO: 13 736 of SEQ ID NO:13 736 of SEQ ID NO: 13

In some embodiments, an AAV Clade F capsid comprises a VP1, VP2, or VP3protein that has at least 85% (e.g., at least 86%, at least 88%, atleast 90%, at least 92%, at least 94%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100%) amino acid sequence identity to aminoacids 1 to 736, amino acids 138 to 736 or amino acids 203 to 736 of SEQID NO:1, respectively, which correspond to the amino acid sequences ofAAV9 capsid proteins VP1, VP2 and VP3, respectively. In someembodiments, an AAV Clade F capsid comprises VP1 and VP2 proteins thathave at least 85% (e.g., at least 86%, at least 88%, at least 90%, atleast 92%, at least 94%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100%) amino acid sequence identity to amino acids 1 to 736and amino acids 138 to 736 of SEQ ID NO: 1, respectively, whichcorrespond to the amino acid sequences of AAV9 capsid proteins VP1 andVP2, respectively; VP1 and VP3 proteins that have at least 85% (e.g., atleast 86%, at least 88%, at least 90%, at least 92%, at least 94%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100%) amino acidsequence identity to amino acids 1 to 736 and amino acids 203 to 736 ofSEQ ID NO:1, respectively, which correspond to the amino acid sequencesof AAV9 capsid proteins VP1 and VP3, respectively; or VP2 and VP3proteins that have at least 85% (e.g., at least 86%, at least 88%, atleast 90%, at least 92%, at least 94%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100%) amino acid sequence identity to aminoacids 138 to 736 and amino acids 203 to 736 of SEQ ID NO:1,respectively, which correspond to the amino acid sequences of AAV9capsid proteins VP2 and VP3, respectively. In some embodiments, an AAVClade F capsid comprises VP1, VP2, and VP3 proteins that have at least85% (e.g., at least 86%, at least 88%, at least 90%, at least 92%, atleast 94%, at least 96%, at least 97%, at least 98%, at least 99%, or100%) amino acid sequence identity to amino acids 1 to 736, amino acids138 to 736 and amino acids 203 to 736 of SEQ ID NO:1, respectively,which correspond to the amino acid sequences of AAV9 capsid proteinsVP1, VP2 and VP3, respectively.

In some embodiments, an AAV Clade F capsid comprises a VP1, VP2, or VP3protein that has at least 85% (e.g., at least 86%, at least 88%, atleast 90%, at least 92%, at least 94%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100%) amino acid sequence identity to aminoacids 1 to 736, amino acids 138 to 736 or amino acids 203 to 736 of anyone of SEQ ID NOs: 2, 3, 5, 6, 11, 7, 8, 9, 10, 4, 12, 14, 15, 16, 17 or13, respectively, which correspond to the amino acid sequences of AAVF1through AAVF9 and AAVF11 through AAVF17 capsid proteins VP1, VP2 andVP3, respectively. In some embodiments, an AAV Clade F capsid comprisesVP1 and VP2 proteins that have at least 85% (e.g., at least 86%, atleast 88%, at least 90%, at least 92%, at least 94%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100%) amino acid sequenceidentity to amino acids 1 to 736 and amino acids 138 to 736 of any oneof SEQ ID NOs: 2, 3, 5, 6, 11, 7, 8, 9, 10, 4, 12, 14, 15, 16, 17 or 13,respectively, which correspond to the amino acid sequences of AAVF1through AAVF9 and AAVF11 through AAVF17 capsid proteins VP1 and VP2,respectively; VP1 and VP3 proteins that have at least 85% (e.g., atleast 86%, at least 88%, at least 90%, at least 92%, at least 94%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100%) amino acidsequence identity to amino acids 1 to 736 and amino acids 203 to 736 ofany one of SEQ ID NOs: 2, 3, 5, 6, 11, 7, 8, 9, 10, 4, 12, 14, 15, 16,17 or 13, respectively, which correspond to the amino acid sequences ofAAVF1 through AAVF9 and AAVF11 through AAVF17 capsid proteins VP1 andVP3, respectively; or VP2 and VP3 proteins that have at least 85% (e.g.,at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, atleast 96%, at least 97%, at least 98%, at least 99%, or 100%) amino acidsequence identity to amino acids 138 to 736 and amino acids 203 to 736of any one of SEQ ID NOs: 2, 3, 5, 6, 11, 7, 8, 9, 10, 4, 12, 14, 15,16, 17 or 13, respectively, which correspond to the amino acid sequencesof AAVF1 through AAVF9 and AAVF11 through AAVF17 capsid proteins VP2 andVP3, respectively. In some embodiments, an AAV Clade F capsid comprisesVP1, VP2, and VP3 proteins that have at least 85% (e.g., at least 86%,at least 88%, at least 90%, at least 92%, at least 94%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100%) amino acid sequenceidentity to amino acids 1 to 736, amino acids 138 to 736 and amino acids203 to 736 of any one of SEQ ID NOs: 2, 3, 5, 6, 11, 7, 8, 9, 10, 4, 12,14, 15, 16, 17 or 13, respectively, which correspond to the amino acidsequences of AAVF1 through AAVF9 and AAVF11 through AAVF17 capsidproteins VP1, VP2 and VP3, respectively.

In some embodiments, an AAV Clade F capsid comprises a VP1, VP2, or VP3protein that is encoded by a nucleotide sequence comprising at least 85%(e.g., at least 86%, at least 88%, at least 90%, at least 92%, at least94%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%)nucleotide sequence identity to SEQ ID NO:18, which corresponds to thenucleotide sequence encoding AAV9 capsid proteins VP1, VP2 and VP3,respectively. In some embodiments, an AAV Clade F capsid comprises VP1and VP2 proteins that are encoded by nucleotide sequences comprising atleast 85% (e.g., at least 86%, at least 88%, at least 90%, at least 92%,at least 94%, at least 96%, at least 97%, at least 98%, at least 99%, or100%) nucleotide sequence identity to SEQ ID NO: 18, which correspondsto the nucleotide sequence encoding AAV9 capsid proteins VP1, VP2, andVP3; VP1 and VP3 proteins that are encoded by a nucleotide sequencecomprising at least 85% (e.g., at least 86%, at least 88%, at least 90%,at least 92%, at least 94%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100%) nucleotide sequence identity to SEQ ID NO: 18; orVP2 and VP3 proteins that are encoded by a nucleotide sequencecomprising at least 85% (e.g., at least 86%, at least 88%, at least 90%,at least 92%, at least 94%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100%) nucleotide sequence identity to SEQ ID NO: 18. Insome embodiments, an AAV Clade F capsid comprises VP1, VP2, and VP3proteins that are encoded by a nucleotide sequence comprising at least85% (e.g., at least 86%, at least 88%, at least 90%, at least 92%, atleast 94%, at least 96%, at least 97%, at least 98%, at least 99%, or100%) nucleotide sequence identity to SEQ ID NO: 18, which correspondsto a nucleotide sequence encoding AAV9 capsid proteins VP1, VP2 and VP3.

In some embodiments, an AAV Clade F capsid comprises a VP1, VP2, or VP3protein that is encoded by a nucleotide sequence comprising at least 85%(e.g., at least 86%, at least 88%, at least 90%, at least 92%, at least94%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%)nucleotide sequence identity to any one of SEQ ID NOs: 20, 21, 22, 23,25, 24, 27, 28, 29, 26, 30, 31, 32, 33, 34 or 35, which correspond tonucleotide sequences encoding AAVF1 through AAVF17 capsid proteins VP1,VP2 and VP3, respectively. In some embodiments, an AAV Clade F capsidcomprises VP1 and VP2 proteins that are encoded by nucleotide sequencescomprising at least 85% (e.g., at least 86%, at least 88%, at least 90%,at least 92%, at least 94%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100%) nucleotide sequence identity to any one of SEQ IDNOs:20-35; VP1 and VP3 proteins that are encoded by a nucleotidesequence comprising at least 85% (e.g., at least 86%, at least 88%, atleast 90%, at least 92%, at least 94%, at least 96%, at least 97%, atleast 98%, at least 99%, or 100%) nucleotide sequence identity to anyone of SEQ ID NOs:20-35; or VP2 and VP3 proteins that are encoded by anucleotide sequence comprising at least 85% (e.g., at least 86%, atleast 88%, at least 90%, at least 92%, at least 94%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100%) nucleotide sequenceidentity to any one of SEQ ID NOs:20-35. In some embodiments, an AAVClade F capsid comprises VP1, VP2, and VP3 proteins that are encoded bya nucleotide sequence comprising at least 85% (e.g., at least 86%, atleast 88%, at least 90%, at least 92%, at least 94%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100%) nucleotide sequenceidentity to any one of SEQ ID NOs: 20, 21, 22, 23, 25, 24, 27, 28, 29,26, 30, 31, 32, 33, 34 or 35, which correspond to nucleotide sequencesencoding AAVF1 through AAVF17 capsid proteins VP1, VP2 and VP3,respectively.

In some embodiments, an AAV Clade F capsid comprises an AAV9 VP1, VP2,or VP3 capsid protein, which corresponds to amino acids 1 to 736, aminoacids 138 to 736 and amino acids 203 to 736 as set forth in SEQ ID NO:1,respectively. In some embodiments, an AAV Clade F capsid comprises AAV9VP1 and VP2 capsid proteins, which correspond to amino acids 1 to 736and amino acids 138 to 736 as set forth in SEQ ID NO:1, respectively;AAV9 VP1 and VP3 capsid proteins, which correspond to amino acids 1 to736 and amino acids 203 to 736 as set forth in SEQ ID NO: 1,respectively; or AAV9 VP2 and VP3 capsid proteins, which correspond toamino acids 138 to 736 and amino acids 203 to 736 as set forth in SEQ IDNO:1, respectively. In some embodiments, an AAV Clade F capsid comprisesAAV9 VP1, VP2 and VP3 capsid proteins, which correspond to amino acids 1to 736, amino acids 138 to 736 and amino acids 203 to 736 as set forthin SEQ ID NO:1, respectively.

In some embodiments, an AAV Clade F capsid comprises a VP1 capsidprotein selected from a VP1 capsid protein of any one of AAVF1 throughAAVF9 and AAVF11 through AAVF17, which corresponds to amino acids 1 to736 as set forth in SEQ ID NOs: 2, 3, 5, 6, 11, 7, 8, 9, 10, 4, 12, 14,15, 16, 17 or 13, respectively. In some embodiments, an AAV Clade Fcapsid comprises a VP1 and a VP2 capsid protein independently selectedfrom a VP1 and VP2 capsid protein of any one of AAVF1 through AAVF9 andAAVF11 through AAVF17, which correspond to amino acids 1 to 736 andamino acids 138 to 736 as set forth in SEQ ID NOs: 2, 3, 5, 6, 11, 7, 8,9, 10, 4, 12, 14, 15, 16, 17 or 13, respectively. In some embodiments,an AAV Clade F capsid comprises a VP2 and a VP3 capsid proteinindependently selected from a VP2 and VP3 capsid protein of any one ofAAVF1 through AAVF9 and AAVF11 through AAVF17, which correspond to aminoacids 138 to 736 and amino acids 203 to 736 as set forth in SEQ ID NOs:2, 3, 5, 6, 11, 7, 8, 9, 10, 4, 12, 14, 15, 16, 17 or 13, respectively.In some embodiments, an AAV Clade F capsid comprises each of the VP1,VP2 and VP3 capsid proteins of any one of AAVF1 through AAVF9 and AAVF11through AAVF17, which correspond to amino acids 1 to 736, amino acids138 to 736 and amino acids 203 to 736 as set forth in SEQ ID NOs: 2, 3,5, 6, 11, 7, 8, 9, 10, 4, 12, 14, 15, 16, 17 or 13, respectively.

As used herein, a fragment of a polynucleotide sequence may be a portionof the polynucleotide that encodes a polypeptide which providessubstantially the same function as the polypeptide encoded by the fulllength polynucleotide sequence. As used herein, mutants of apolynucleotide sequence may be obtained by deletion, substitution,addition, and/or insertion of one or more nucleotides to the specificpolynucleotide sequence. It should be understood that such fragments,and/or mutants of a polynucleotide sequence encode a polypeptide havingsubstantially the same function as the polypeptide encoded by the fulllength polynucleotide sequence.

As used herein, a polypeptide sequence may include fragments, and/ormutants of the polypeptide sequence, while still providing substantiallythe same function as the full length polypeptide sequence. A fragment ofa polypeptide sequence means a part of the polypeptide sequence thatprovides substantially the same function as the full length polypeptidesequence. Examples of mutants of a polypeptide sequence includedeletions, substitutions, additions, and/or insertions of one or moreamino acids to the polypeptide sequence.

In certain embodiments, a polynucleotide sequence may be a recombinantor non-naturally occurring polynucleotide. In certain embodiments, apolynucleotide sequence may be cDNA.

In certain embodiments, the AAV Clade F vectors or AAV vector variantsprovided herein may comprise any of the AAVF (or AAVHSC) or any otherAAV Clade F vectors described herein. In certain embodiments, a AAVClade F vector or AAV vector variant may comprise any of the AAVF (orAAVHSC) vectors described in herein, such as AAVF1, AAVF2, AAVF3, AAVF4,AAVF5, AAVF6, AAVF7, AAVF8, AAVF9, AAVF10, AAVF11, AAVF12, AAVF13,AAVF14, AAVF15, AAVF16, AAVF17, variants, fragments, mutants, or anycombination thereof. In certain embodiments, a Clade F vector or AAVvector variant may comprise any of AAV9, AAVF1, AAVF2, AAVF3, AAVF4,AAVF5, AAVF6, AAVF7, AAVF8, AAVF9, AAVF10, AAVF11, AAVF12, AAVF13,AAVF14, AAVF15, AAVF16, AAVF17, AAVHU31, AAVHU32, variants, fragments,mutants, or any combination thereof.

In certain embodiments, the AAV Clade F vectors or AAV vector variantsprovided herein may comprise an editing element (also referred to hereinas a targeting cassette, meaning the terms “editing element” and“targeting cassette” are used interchangeably herein) comprising one ormore nucleotide sequences or an internucleotide bond to be integratedinto a target locus (also referred to herein as a target site, meaningthe terms are used interchangeably herein) of the genome, a 5′homologous arm nucleotide sequence 5′ of the editing element, havinghomology to a 5′ region of the mammalian chromosome relative to thetarget locus (e.g., a 5′ homologous arm polynucleotide sequence flankingthe editing element (targeting cassette) and having homology to a regionthat is upstream of the target locus (target site)), and a 3′ homologousarm nucleotide sequence 3′ of the editing element, having homology to a3′ region of the mammalian chromosome relative to the target locus(e.g., a 3′ homologous arm polynucleotide sequence flanking the editingelement (targeting cassette) and having homology to a region that isdownstream of the target locus (target site)).

In certain embodiments, the one or more nucleotide sequences to beintegrated into a target site of the genome may be one or moretherapeutic nucleotide sequences. The term “therapeutic” as used hereinrefers to a substance or process that results in the treatment of adisease or disorder. “Therapeutic nucleotide sequence” is a nucleotidesequence that provides a therapeutic effect. The therapeutic effect canbe direct (e.g., substitution of a nucleic acid of a gene expressed as aprotein, or insertion of a cDNA into an intron for expression) orindirect (e.g., correction of a regulatory element such as a promoter).In certain embodiments, the therapeutic nucleotide sequence may includeone or more nucleotides. In certain embodiments, the therapeuticnucleotide sequence may be a gene, variant, fragment, or mutant thereof.In certain embodiments, when gene therapy is desired, the therapeuticnucleotide sequence may be any nucleotide sequence that encodes aprotein that is therapeutically effective, including therapeuticantibodies. The Clade F vectors or AAV vector variants comprising thetherapeutic nucleotide sequences are preferably administered in atherapeutically effective amount via a suitable route of administration,such as injection, inhalation, absorption, ingestion or other methods.

In some embodiments, an editing element as described herein consists ofone nucleotide. In some embodiments, an editing element as describedherein consists of one nucleotide and a target locus as described hereinis a nucleotide sequence consisting of one nucleotide, the target locusrepresenting a point mutation. In some embodiments, an editing elementas described herein comprises at least 1, 2, 10, 100, 200, 500, 1000,1500, 2000, 3000, 4000, or 5000 nucleotides. In some embodiments, anediting element as described herein comprises or consists of 1 to 5500,1 to 5000, 1 to 4500, 1 to 4000, 1 to 3000, 1 to 2000, 1 to 1000, 1 to500, 1 to 200, or 1 to 100 nucleotides, or 2 to 5500, 2 to 5000, 2 to4500, 2 to 4000, 2 to 3000, 2 to 2000, 2 to 1000, 2 to 500, 2 to 200, or2 to 100 nucleotides, or 10 to 5500, 10 to 5000, 10 to 4500, 10 to 4000,10 to 3000, 10 to 2000, 10 to 1000, 10 to 500, 10 to 200, or 10 to 100nucleotides. In some embodiments, an editing element as described hereincomprises or consists of an exon, an intron, a 5′ untranslated region(UTR), a 3′ UTR, a promoter, a splice donor, a splice acceptor, asequence encoding or non-coding RNA, an insulator, a gene, or acombination thereof. In some embodiments, an editing element asdescribed herein is a fragment (e.g., no more than 2 kb, no more than 1kb, no more than 500 bp, no more than 250 bp, no more than 100 bp, nomore than 50 bp, or no more than 25 bp) of a coding sequence of a genewithin or spanning a target locus as described herein. In someembodiments, an editing element as described herein is aninternucleotide bond (e.g., a phosphodiester bond connecting twoadjacent nucleotides). In some embodiments, an editing element asdescribed herein is an internucleotide bond, a target locus in achromosome as described herein is a nucleotide sequence comprising oneor more nucleotides, and the editing element comprises a deletion forthe target locus in the chromosome.

In certain embodiments, the editing element (or targeting cassette) ofthe AAV Clade F vector or AAV vector variant may comprise one or moreregulatory element polynucleotide sequences. For example, in certainembodiments, the one or more regulatory element polynucleotide sequencesmay be selected from a 2A sequence, splice acceptor sequence,polyadenylation sequence, and any combination thereof. In certainembodiments, the targeting cassette may comprise one or more AAVinverted terminal repeat (ITR) polynucleotide sequences flanking the 5′and 3′ homologous arm polynucleotide sequences. In certain embodiments,the editing element (or targeting cassette) does not contain a promoterto drive expression of the one or more nucleotide sequences. In certainembodiments, if the editing element (or targeting cassette) does notcontain a promoter, the expression of the one or more nucleotidesequences after integration into the cell genome may be controlled byone or more regulatory elements of the cell. In certain embodiments,expression of the promoterless one or more nucleotide sequencesdemonstrates that the one or more nucleotide sequences was correctlyintegrated into the cell.

In certain embodiments, the AAV Clade F vector or AAV vector variant maycomprise one or more homologous arm polynucleotide sequences. In certainembodiments, the one or more homologous arm polynucleotide sequences maybe homologous to a region of the target locus (target site) of thegenome. In certain embodiments, the one or more homologous armpolynucleotide sequences may be a 5′ homologous arm polynucleotidesequence. In certain embodiments, the 5′ homologous arm polynucleotidesequence may flank the 5′ end of the editing element (or targetingcassette). In certain embodiments, the 5′ homologous arm polynucleotidesequence flanking the editing element (or targeting cassette) may behomologous to a region that is upstream of a target locus (target site)of the genome. In certain embodiments, the one or more homologous armpolynucleotide sequences may be a 3′ homologous arm polynucleotidesequence. In certain embodiments, the 3′ homologous arm polynucleotidesequence may flank the 3′ end of the editing element (or targetingcassette). In certain embodiments, the 3′ homologous arm polynucleotidesequence flanking the editing element (or targeting cassette) may behomologous to a region that is downstream of the target locus (targetsite) of the genome. In certain embodiments, the homologous armpolynucleotide sequences may be approximately 500 to 1,000 nucleotideslong. For example, in certain embodiments, the homologous armpolynucleotide sequences may be approximately 800 nucleotides long. Incertain embodiments, the homologous arm polynucleotide sequence may beup to approximately 3,000 nucleotides long. In some embodiments, each ofthe 5′ and 3′ homologous arm nucleotide sequences independently has anucleotide length of between about 50 to 2000 nucleotides, such asbetween about 500-1000, about 600-1000, or about 700-900 nucleotides. Insome embodiments, each of the 5′ and 3′ homologous arm nucleotidesequences independently has a nucleotide length of between about 600,about 800, or about 1000 nucleotides.

In some embodiments, the 5′ and 3′ homologous arm nucleotide sequenceshave substantially equal nucleotide lengths. In some embodiments, the 5′and 3′ homologous arm nucleotide sequences have asymmetrical nucleotidelengths. In some embodiments, the asymmetry in nucleotide length isdefined by a difference between the 5′ and 3′ homologous arm nucleotidesequence lengths of up to 50% in the length, such as up to 40%, 30%,20%, or 10% difference in the length. In some embodiments, the asymmetryin nucleotide length is defined by on arm of the 5′ and 3′ homologousarm having a length of about 600 nucleotides and the other arm of the 5′and 3′ homologous arm having a length of about 800 or about 900nucleotides.

In some embodiments, the 5′ homologous arm nucleotide sequence has atleast about 90% (e.g., at least about 90%, at least about 95%, at leastabout 96%, at least about 97%, at least about 98%, at least about 99%,or at least about 99.5%) nucleotide sequence identity to the 5′ regionof the mammalian chromosome relative to the target locus. In someembodiments, the 3′ homologous arm nucleotide sequence has at leastabout 90% (e.g., at least about 90%, at least about 95%, at least about96%, at least about 97%, at least about 98%, at least about 99%, or atleast about 99.5%) nucleotide sequence identity to the 3′ region of themammalian chromosome relative to the target locus. In some embodiments,differences in nucleotide sequences of the 5′ homologous arm or the 3′homologous arm and the corresponding 5′ region or 3′ region of themammalian chromosome, respectively, can comprise, consist essentially ofor consist of non-coding differences in nucleotide sequences. In someembodiments, differences in nucleotide sequences of the 5′ homologousarm or the 3′ homologous arm and the corresponding 5′ region or 3′region of the mammalian chromosome, respectively, can comprise, consistessentially of or consist of differences in nucleotide sequences thatresult in conservative amino acid changes (e.g., a basic amino acidchanged to a different basic amino acid). In some embodiments, the 5′homologous arm nucleotide sequence has 100% sequence identity to the 5′region of the mammalian chromosome relative to the target locus and the3′ homologous arm nucleotide sequence has 100% sequence identity to the3′ region of the mammalian chromosome relative to the target locus. Insome embodiments, the 5′ homologous arm nucleotide sequence and the 3′homologous arm nucleotide sequence are considered homologous with the 5′region and 3′ of the mammalian chromosome relative to the target locus,respectively, even if the target locus contains one or more mutations,such as one or more naturally occurring SNPs, compared to the 5′ or 3′homologous arm.

In certain embodiments, the target locus (target site) of the cellgenome may be any region of the genome where it is desired that theediting of the cell genome occur. For example, the target locus (targetsite) of the cell genome may comprise a locus of a chromosome in thecell (e.g., a region of a mammalian chromosome). In certain embodiments,the locus of the chromosome may be a safe harbor site. A safe harborsite is a location in the genome where a nucleotide sequence mayintegrate and function in a predictable manner without perturbingendogenous gene activity. In certain embodiments, the safe harbor sitemay be the AAVS1 locus in human chromosome 19 (also known as PPP1R12Clocus). In certain embodiments, the safe harbor site may be the firstintron of PPP1R12C in the AAVS1 locus in human chromosome 19. The AAVS1locus on chromosome 19 qter13.3-13.4 was previously shown to be a “safeharbor” site for the insertion of transgenes since genes inserted hereare expressed with no pathogenic consequences, which is similar towild-type AAV that integrates at this locus with no pathogenicconsequences (Giraud 1994; Linden, 1996A; Linden 1996B). In someembodiments, the target locus (target site) is a locus associated with adisease state as described herein.

In certain embodiments, the target locus is a mutant target locus in amammalian chromosome comprising one or more mutant nucleotides, relativeto a corresponding wild type mammalian chromosome. In some embodiments,the mutant target locus comprises a point mutation, a missense mutation,a nonsense mutation, an insertion of one or more nucleotides, a deletionof one or more nucleotides, or combinations thereof. In someembodiments, the mutant target locus comprises an amorphic mutation, aneomorphic mutation, or an antimorphic mutation. In some embodiments,the mutant target locus comprises an autosomal dominant mutation, anautosomal recessive mutation, a heterozygous mutation, a homozygousmutation, or combinations thereof. In some embodiments of any one of themutant target loci described herein, the mutant target locus is selectedfrom a promoter, an enhancer, a signal sequence, an intron, an exon, asplice donor site, a splice acceptor site, an internal ribosome entrysite, an inverted exon, an insulator, a gene, a chromosomal inversion,and a chromosomal translocation within the mammalian chromosome.

In some embodiments, a target locus in a chromosome as described hereinis a nucleotide sequence comprising n nucleotides where n is an integergreater than or equal to one (e.g., 1, 2, 3, 4, 5, 10, 20, 30, 40, 50,100, 500, 1000, 2000, 3000, 4000. 5000, or any integer therebetween), anediting element as described herein comprises m nucleotides where m isan integer equal to n, and the editing element represents a substitutionfor the target locus of the chromosome. In some embodiments, a targetlocus in a chromosome as described herein is a nucleotide sequencecomprising n nucleotides where n is an integer greater than or equal toone (e.g., 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 100, 500, 1000, 2000,3000, 4000. 5000, or any integer therebetween), an editing element asdescribed herein comprises m nucleotides where m is an integer greaterthan n, and the editing element represents a substitutive addition forthe target locus of the chromosome. In some embodiments, a target locusin a chromosome as described herein is a nucleotide sequence comprisingn nucleotides where n is an integer greater than or equal to two (e.g.,2, 3, 4, 5, 10, 20, 30, 40, 50, 100, 500, 1000, 2000, 3000, 4000. 5000,or any integer therebetween), an editing element as described hereincomprises m nucleotides where m is an integer less than n; and theediting element represents a substitutive deletion for the target locusof the chromosome. In some embodiments, a target locus in a chromosomeas described herein is an internucleotide bond, an editing element asdescribed herein comprises m nucleotides where m is an integer greaterthan or equal to one (e.g., 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 100, 500,1000, 2000, 3000, 4000. 5000, or any integer therebetween); and theediting element represents an addition for the target locus of thechromosome.

In some embodiments, a target locus in a chromosome is a target locus ina mammalian chromosome (e.g. a human, mouse, bovine, equine, canine,feline, rat, or rabbit chromosome). In some embodiments, the targetlocus can comprise an intron of a mammalian chromosome. In someembodiments, the target locus can comprise an exon of a mammalianchromosome. In some embodiments, the target locus can comprise anon-coding region of a mammalian chromosome. In some embodiments, thetarget locus can comprise a regulatory region of a mammalian chromosome.In some embodiments, the mammalian chromosome is selected from humanchromosome 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, X and Y. In some embodiments, the mammalianchromosome is selected from mouse chromosome 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, X and Y. In some embodiments,the mammalian chromosome is not human chromosome 19. In someembodiments, the mammalian chromosome is a somatic cell chromosome.Exemplary somatic cells are further described herein.

In certain embodiments, the one or more nucleotide sequences or editingelement may be integrated into the genome through homologousrecombination without the need for DNA cleavage prior to integration. Incertain embodiments, the one or more nucleotide sequences or editingelement may be integrated into the genome through homologousrecombination without the need for the addition of exogenous nucleasessuch as a zinc finger nuclease (ZFN), a transcription activator-likeeffector nuclease (TALEN), or an RNA guided nuclease (CRISPR/Cas).

In certain embodiments, the cell that is edited by the AAV Clade Fvectors or AAV vector variants described herein may be any type of cell.In certain embodiments, the cell may be a wide variety of mammaliancells, for example, cells of the liver, lung, cartilage and otherconnective tissue, eye, central and peripheral nervous system, lymphaticsystem, bone, muscle, blood, brain, skin, heart, and digestive tract.When the cell to be edited by the AAV Clade F vectors or AAV vectorvariants is, for example, a liver cell, the inserted nucleotide sequenceis directed to treating (improving or curing a disorder or stoppingfurther progression of a disease or disorder) or preventing a condition.When the cell to be edited by the AAV Clade F vectors or AAV vectorvariants is a liver cell, the liver conditions treated or preventedcomprise hemophilia, enzyme delivery, cirrhosis, cancer, oratherosclerosis, among other liver conditions. In certain embodiments,the cell may be a somatic cell (e.g., a mammalian somatic cell). Incertain embodiments, the cell (e.g., somatic cell such as mammaliansomatic cell) may be from a tissue selected from the group consisting ofconnective tissue (including blood), muscle tissue, nervous tissue, andepithelial tissue. In certain embodiments, the cell (e.g., somatic cellsuch as mammalian somatic cell) may be from an organ selected from thegroup consisting of lung, heart, liver, kidney, muscle, brain, eye,breast, bone, and cartilage. In some embodiments, the cell is a CD34+cell (e.g., a CD34+ somatic cell). In some embodiments, the cell (e.g.,somatic cell such as mammalian somatic cell) is a liver cell, afibroblast, a breast cell, a lymphocyte, or a retinal cell.

As shown herein, AAV packaged with the Clade F capsids or capsidvariants described herein demonstrate specific tropism for certaintarget tissues, such as blood stem cells, liver, heart, eye, breast, andjoint tissue, and may be used to transduce stem cells for introductionof genes of interest into the target tissues. Certain of the vectors areable to cross tightly controlled biological junctions, such as theblood-brain barrier, which open up additional novel uses and targetorgans for the vectors, providing for methods of gene therapy throughgenome editing. Thus, Clade F vectors or AAV vector variants maydemonstrate a tropism for a particular cell based on their Clade Fcapsids or capsid variants. For example a) for muscle tissue or cells,the AAV Clade F vector or AAV vector variant may be selected from thegroup of AAVF5, AAVF7, AAVF13, AAVF15, and AAVF17; b) for heart or lungtissue or cells, the vector may be selected from the group of AAVF13,AAVF15, and AAVF17; c) for liver or CNS tissue or cells, the vector maybe selected from AAVF5, AAVF13, AAVF17, AAVF7 or AAVF15; d) for stemcells, the vector may be AAVF17; e) for B cell progenitors, the vectormay be AAVF5; f) for myeloid and erythroid progenitors, the vector maybe AAVF12; and g) for lymph node, kidney, spleen, cartilage and bonetissues or cells, the vector may be selected from the group of thevector selected from the group of AAVF7, AAVF13, AAVF15, and AAVF17.

In addition, Clade F vectors or AAV vector variants may have a tropismfor cells containing various tags, such as a six-His tag or an affinitytag, or for interferon responses, such as naturally occurring antibodieselicited or introduced monoclonal antibodies administered in response toa pathogen or tumor cell.

In certain embodiments, the cell may be a stem cell (e.g., a mammalianstem cell). In certain embodiments, the stem cell may be any type ofstem cell including a hematopoietic stem cell, a pluripotent stem cell,an embryonic stem cell or a mesenchymal stem cell. In certainembodiments, the stem cell (e.g., mammalian stem cell) may be ahematopoietic stem cell, a cord blood stem cell, a bone marrow stemcell, a fetal liver stem cell, or a peripheral blood stem cell. In someembodiments, the stem cell may be a CD34+ stem cell. In certainembodiments, the stem cell (e.g., mammalian stem cell) may be ahematopoietic stem cell or peripheral blood stem cell. Transduction ofthe stem cell may be either transient or permanent (also calledpersistent). If transient, one embodiment allows for the length of timethe therapeutic nucleotide is used or expressed to be controlled eitherby the vector, by substance attached to the vector, or by externalfactors or forces.

In certain embodiments, the cell may be selected from the groupconsisting of a CD34+ Hematopoietic stem cell line (HSC), a K562 CD34+leukemia cell line, a HepG2 human liver cell line, a peripheral bloodstem cell, a cord blood stem cell, a CD34+ peripheral blood stem cell, aWI-38 human diploid fibroblast cell line, a MCF7 human breast cancercell line, a Y79 human retinoblastoma cell line, a SCID-X1 LBL humanEBV-immortalized B cell line, a primary human hepatocyte, a primaryhepatic sinusoidal endothelial cell, and a primary skeletal musclemyoblast.

Also provided herein are methods of ex-vivo editing a genome of a cellof a subject comprising transducing the cell with a Clade F or an AAVvector variant as described herein. In certain embodiments, transducingthe cell with a Clade F vector or an AAV vector variant may occurwithout additional exogenous nucleases, such as a zinc finger nuclease(ZFN), a transcription activator-like effector nuclease (TALEN), or anRNA guided nuclease (CRISPR/Cas). In certain embodiments, the cell maybe any type of cell. In certain embodiments, the cell may be a stem cellas described herein. For example, in certain embodiments, methods ofediting the genome of a stem cell may comprise transducing the stem cellwith one or more Clade F vectors or AAV vector variants. In certainembodiments, transduction of the stem cell may be performed without theneed for additional exogenous nucleases. In certain embodiments, thecell may be a somatic cell as described herein. For example, in certainembodiments, methods of editing the genome of a somatic cell maycomprise transducing the somatic cell with one or more Clade F vectorsor AAV vector variants. In certain embodiments, transduction of thesomatic cell may be performed without the need for additional exogenousnucleases. In certain embodiments, the Clade F vector or AAV vectorvariant comprises one or more Clade F capsids or capsid variants(variant relative to AAV9), an editing element (targeting cassette)selected from an internucleotide bond or a nucleotide sequence forintegration into a target locus of a mammalian chromosome or comprisingone or more therapeutic nucleotide sequences to be integrated into atarget locus (target site) of the genome, a 5′ homologous armpolynucleotide sequence flanking the editing element (targetingcassette) and having homology to a region that is upstream of the targetlocus (target site), and a 3′ homologous arm polynucleotide sequenceflanking the editing element (targeting cassette) and having homology toa region that is downstream of the target locus (target site). Incertain embodiments, the internucleotide bond or nucleotide sequence orthe one or more therapeutic nucleotide sequences may be integrated intothe genome without the need for additional exogenous nucleases for DNAcleavage prior to integration.

Also provided herein are methods of treating a disease or disorder in asubject by ex-vivo editing a genome of a cell of the subject includingtransducing the cell with a Clade F vector or an AAV vector variant andfurther transplanting the transduced cell into the subject to treat thedisease or disorder. In certain embodiments, the method may comprisetransducing the cell of the subject with a Clade F vector or an AAVvector variant vector described herein. In certain embodiments, the cellmay be transduced without additional exogenous nucleases. In certainembodiments, transduction of the cell with the Clade F vector or the AAVvector variant may be performed as provided herein or by any method oftransduction known to one of ordinary skill in the art. In certainembodiments, the cell may be transduced with the Clade F vector or theAAV vector variant at a multiplicity of infection (MOI) of 50,000;100,000; 150,000; 200,000; 250,000; 300,000; 350,000; 400,000; 450,000;or 500,000, or at any MOI that provides for optimal transduction of thecell. In certain embodiments, the transduced cell is furthertransplanted into the subject, wherein the transduced cell treats thedisease or disorder. In certain embodiments, the cell may be any type ofcell described herein.

Also provided herein are methods of editing a target locus of amammalian genome as described herein. In some embodiments, the methodcomprises transducing a cell (such as a human, mouse, bovine, equine,canine, feline, rat, or rabbit cell) comprising the mammalian genomewith an AAV as described herein (e.g., a replication-defective AAVcomprising a correction genome enclosed in a capsid). In someembodiments, the method comprises (a) obtaining mammalian cells from amammal (such as a human, mouse, bovine, equine, canine, feline, rat, orrabbit); (b) culturing the mammalian cells ex-vivo to form an ex-vivoculture; (c) transducing the mammalian cells with an AAV as describedherein (e.g., a replication-defective AAV comprising a correction genomeenclosed in a capsid) in the ex-vivo culture to form transducedmammalian cells; and (d) administering the transduced mammalian cells tothe mammal. In some embodiments, the method comprises (a) obtainingmammalian cells from a first mammal; (b) culturing the mammalian cellsex-vivo to form an ex-vivo culture; (c) transducing the mammalian cellswith an AAV as described herein (e.g., a replication-defective AAVcomprising a correction genome enclosed in a capsid in the ex-vivoculture to form transduced mammalian cells; and (d) administering thetransduced mammalian cells to a second mammal. In some embodiments, thefirst mammal and the second mammal are different species (e.g., thefirst mammal is human, mouse, bovine, equine, canine, feline, rat, orrabbit and the second mammal is a different species). In someembodiments, the first mammal and the second mammal are the same species(e.g., are both human, mouse, bovine, equine, canine, feline, rat, orrabbit). In some embodiments, the method comprises administering an AAVas described herein (e.g., a replication-defective AAV comprising acorrection genome enclosed in a capsid) to a mammal (such as a human,mouse, bovine, equine, canine, feline, rat, or rabbit) in an amounteffective to transduce cells of the mammal with the AAV in-vivo.

In some embodiments of any one of the methods, the mammalian cells arefrom a tissue selected from the group consisting of connective tissue(including blood), muscle tissue, nervous tissue, and epithelial tissue.In some embodiments of any one of the methods, the mammalian cells arefrom an organ selected from the group consisting of lung, heart, liver,kidney, muscle, brain, eye, breast, bone, and cartilage. In someembodiments of any one of the methods, the mammalian cells are stemcells. In some embodiments, the stem cells are hematopoietic stem cellsor peripheral blood stem cells. In some embodiments of any one of themethods, the mammalian cells are a CD34+ cells.

In some embodiments of any one of the methods, the AAV (e.g., Clade FAAV) is transduced or administered without co-transducing orco-administering an exogenous nuclease or a nucleotide sequence thatencodes an exogenous nuclease. Exemplary exogenous nucleases include azinc finger nuclease (ZFN), a transcription activator-like effectornuclease (TALEN), or an RNA guided nuclease (CRISPR/Cas). In someembodiments of any one of the methods, the AAV is transduced oradministered without co-transducing or co-administering an exogenouszinc finger nuclease or a nucleotide sequence that encodes an exogenouszinc finger nuclease. In some embodiments, the zinc finger nuclease is azinc finger nuclease comprising a DNA-binding domain that targets theAAVS1 locus (e.g., a DNA-binding domain that targets the first intron ofPPP1R12C in the AAVS1 locus).

In some embodiments of any one of the methods, the AAV (e.g., Clade FAAV) has a chromosomal integration efficiency of at least about 1%(e.g., at least about 2%, at least about 3%, at least about 4%, at leastabout 5%, at least about 10%, at least about 20%, at least about 30%, atleast about 40%, at least about 50%, at least about 60%, at least about70%, at least about 80%, at least about 90%, at least about 95%, atleast about 98%, or about 100%) for integrating the editing element intothe target locus of the mammalian chromosome. In some embodiments of anyone of the methods, the AAV (e.g. Clade F AAV) has a chromosomalintegration efficiency of at least about 1% (e.g., at least about 2%, atleast about 3%, at least about 4%, at least about 5%, at least about10%, at least about 20%, at least about 30%, at least about 40%, atleast about 50%, at least about 60%, at least about 70%, at least about80%, at least about 90%, at least about 95%, at least about 98%, orabout 100%) for integrating the editing element into the target locus ofthe mammalian chromosome in the absence of an exogenous nuclease. Insome embodiments of any one of the methods, the editing element of thecorrection genome is integrated into the target locus of the mammalianchromosome with a chromosomal integration efficiency ranging from 10% to70%, 20% to 70%, 40% to 70%, 50% to 70%, 10% to 80%, 20% to 80%, 40% to80%, 50% to 80%, 10% to 90%, 20% to 90%, 40% to 90%, 50% to 90%, 10% to100%, 20% to 100%, 40% to 100%, or 50% to 100% of the mammalian cells.In some embodiments of any one of the methods, the editing element ofthe correction genome is integrated into the target locus of themammalian chromosome with a chromosomal integration efficiency rangingfrom 10% to 70%, 20% to 70%, 40% to 70%, 50% to 70%, 10% to 80%, 20% to80%, 40% to 80%, 50% to 80%, 10% to 90%, 20% to 90%, 40% to 90%, 50% to90%, 10% to 100%, 20% to 100%, 40% to 100%, or 50% to 100% of themammalian cells in the absence of an exogenous nuclease.

In some embodiments of any one of the methods, the AAV (e.g., Clade FAAV) has a chromosomal integration efficiency further characterized byan allele frequency in a population of cells of at least about 10%(e.g., at least about 20%, at least about 30%, at least about 40%, atleast about 50%, at least about 60%, at least about 75%, at least about85%, at least about 90%, or at least about 95%) for the allelecomprising the editing element integrated into the target locus of themammalian chromosome. In some embodiments, the allele frequency in apopulation of cells is an allele frequency in a population of cells invitro, such as population of a cell type provided herein in vitro (e.g.,a CD34+ Hematopoietic stem cell line (HSC), a K562 CD34+ leukemia cellline, a HepG2 human liver cell line, a peripheral blood stem cell, acord blood stem cell, a CD34+ peripheral blood stem cell, a WI-38 humandiploid fibroblast cell line, a MCF7 human breast cancer cell line, aY79 human retinoblastoma cell line, a SCID-X1 LBL human EBV-immortalizedB cell line, a primary human hepatocyte, a primary hepatic sinusoidalendothelial cell, or a primary skeletal muscle myoblast).

According to certain embodiments, methods of treating a disease ordisorder in a subject by ex-vivo editing a genome of stem cell of thesubject and further transplanting the edited cell into the subject totreat the disease or disorder are provided. In certain embodiments, themethods of treating a disease or disorder in a subject by editing agenome of a stem cell of the subject may comprise the steps oftransducing the stem cell of the subject with a AAV Clade F vector or anAAV vector variant as described herein and transplanting the transducedstem cell into the subject, wherein the transduced stem cell treats thedisease or disorder. In certain embodiments, the AAV Clade F vector orthe AAV vector variant may comprise one or more Clade F capsids orcapsid variants, an editing element (targeting cassette) comprising oneor more therapeutic nucleotide sequences to be integrated into a targetlocus (target site) in the genome of the stem cell, a 5′ homologous armpolynucleotide sequence flanking the editing element (targetingcassette) and having homology to a region that is upstream of the targetlocus (target site), and a 3′ homologous arm polynucleotide sequenceflanking the editing element (targeting cassette) and having homology toa region that is downstream of the target locus (target site). Incertain embodiments, transducing the stem cell may be performed withoutadditional exogenous nucleases. In certain embodiments, the one or moretherapeutic nucleotide sequences may be integrated into the genomewithout the need for additional exogenous nucleases for DNA cleavageprior to integration.

In certain embodiments, when the cell is a stem cell, the disease ordisorder that is treated may be any disease or disorder that is causedby one or more mutations of the genome. In certain embodiments, thedisease or disorder that is treated is selected from inherited metabolicdiseases, lysosomal storage diseases, mucopolysaccharidodosis,immunodeficiency diseases, and hemoglobinopathy diseases and infections.In certain embodiments, when the cell to be edited is a stem cell, theAAV Clade F vector or AAV vector variant may be selected from the groupof AAVF7, AAVF12, AAVF15, AAVF17, variants, mutants, and a combinationthereof. In certain embodiments, when the cell to be edited is a stemcell, the Clade F vector or AAV vector variant may be selected from thegroup of AAVF5, AAVF7, AAVF12, AAVF15, AAVF17, variants, mutants, and acombination thereof. In certain embodiments, the Clade F vector or AAVvector variant may comprise one or more Clade F capsids or capsidvariants comprising a polynucleotide sequence selected from the group ofAAVF7 (SEQ ID NO: 27), AAVF12 (SEQ ID NO: 30), AAVF15 (SEQ ID NO: 33),AAVF17 (SEQ ID NO: 35), variants, fragments, mutants and combinationsthereof. In certain embodiments, the Clade F vector or the AAV vectorvariant may comprise one or more Clade F capsids or capsid variantscomprising a polynucleotide sequence selected from the group of AAVF5(SEQ ID NO: 25), AAVF7 (SEQ ID NO: 27), AAVF12 (SEQ ID NO: 30), AAVF15(SEQ ID NO: 33), AAVF17 (SEQ ID NO: 35), variants, fragments, mutantsand combinations thereof. In certain embodiments, the AAV Clade F vectoror the AAV vector variant may comprise one or more Clade F capsids orcapsid variants comprising a polypeptide sequence selected from thegroup of AAVF7 (SEQ ID NO: 8), AAVF12 (SEQ ID NO: 12), AAVF15 (SEQ IDNO: 16), AAVF17 (SEQ ID NO: 13), variants, fragments, mutants andcombinations thereof. In certain embodiments, the AAV Clade F vector orAAV vector variant may comprise one or more Clade F capsids or capsidvariants comprising a polypeptide sequence selected from the group ofAAVF5 (SEQ ID NO: 11), AAVF7 (SEQ ID NO: 8), AAVF12 (SEQ ID NO: 12),AAVF15 (SEQ ID NO: 16), AAVF17 (SEQ ID NO: 13), variants, fragments,mutants and combinations thereof.

In another embodiment, the AAV Clade F vectors or AAV vector variantscapable of genome editing, from CD34+ HSC or from another source, may beused for high efficiency transduction of stem cells, including HSC andiPSC, and other cells, such as those of the heart, joint, centralnervous system, including the brain, muscle, and liver. If the AAV CladeF vectors or AAV vector variants are used in vitro, they may be used forresearch and investigation purposes or to prepare cells or tissues thatwill later be implanted into a subject. Preferably, the subject is amammal, such as a human, but may be any other animal that has tissuesthat can be transduced by the present vectors and methods of using thosevectors. The present AAV Clade F vectors or the AAV vector variants arewell suited for both human and veterinary use. The AAV Clade F vectorsor AAV vector variants may also be used in vitro for the transienttransduction of stem cells, such as HSC. The length of transduction maybe controlled by culture conditions. If the AAV Clade F vectors or AAVvector variants are used in vivo, they may be directly administered tothe subject receiving the therapy for uptake or use in the target cells,such as liver or cartilage cells. If the AAV Clade F vectors or AAVvector variants are used for transducing cells of the central nervoussystem, they are preferably able to traverse the blood-brain barrier andmaintain their efficacy.

Also provided herein are methods of treating a disease or disorder in asubject by in vivo genome editing of a cell of the subject by directlyadministering an AAV Clade F vector or AAV vector variant to thesubject. In certain embodiments, the AAV Clade F vector or AAV vectorvariant may be any AAV Clade F vector or AAV vector variant describedherein. In certain embodiments, the AAV Clade F vector or the AAV vectorvariant may comprise one or more Clade F capsids or capsid variants, anediting element (targeting cassette) comprising one or more therapeuticnucleotide sequences to be integrated into a target locus (target site)in the genome of the stem cell, a 5′ homologous arm polynucleotidesequence flanking the editing element (targeting cassette) and havinghomology to a region that is upstream of the target locus (target site),and a 3′ homologous arm polynucleotide sequence flanking the editingelement (targeting cassette) and having homology to a region that isdownstream of the target locus (target site). In certain embodiments,the AAV Clade F vector or AAV vector variant that is administered treatsthe disease or disorder by genome editing of the cell of the subject. Incertain embodiments, the in vivo genome editing may occur withoutadditional exogenous nucleases. In certain embodiments, the one or moreClade F capsids or capsid variants comprise a polynucleotide orpolypeptide sequence as provided herein. In certain embodiments, thepolynucleotide or polypeptide sequence may be selected from thesequences provided in FIG. 1 of US Patent Publication Number20130096182A1 or in FIG. 1 herein, variants, fragments, mutants, andcombinations thereof. In certain embodiments, the AAV Clade F vectors orAAV vector variants are preferably administered in a therapeuticallyeffective amount via a suitable route of administration, such asinjection, inhalation, absorption, ingestion or other methods.

Previous studies, including Xu et al., Wang et al., and Carbonaro etal., have shown transduction of HSCs following in vivo delivery of aviral vector (see Xu 2004; Wang 2014; and Carbonaro 2006). However, allthree of these studies involved either retroviruses (Xu 2004) orlentiviruses (Wang 2014 and Carbonaro 2006). Additionally, theinjections in Xu et al. and Carbonaro et al. were performed in neonatalmice, and rapamycin and intrafemoral injection was required forefficient transduction in Wang et al. None of these papers, however,report transduction of HSCs by in vivo transduction of Clade F vectorsor AAV vector variants into adult mice. The novel results provided inExample 3 are the first to show AAV vector transduction on HSCs byintravenous injection.

As shown in Example 3 below, intravenous injection of Clade F vectors(or AAV vector variants) pseudotyped with AAVF7 or AAVF17 resulted intransduction of human CD34+ hematopoietic stem and progenitor cells invivo. The intravenous injected Clade F vectors or AAV vectors traffickedto sites of human hematopoiesis and transduced human cells. Intravenousinjection of Clade F vectors or AAV vector variants resulted in Venusexpression in human CD34+ stem progenitor cells as well as their CD45+progeny. These data show that intravenous injection of Clade F vectorsor AAV vector variants can be used for in vivo genome engineeringwithout the need for stem cell harvest, ex vivo transduction,conditioning of the recipient, and subsequent transplantation oftransduced cells. This approach makes stem cell gene therapysignificantly safer, more accessible to patients worldwide, lessexpensive, and obviates the need for hospitalization.

In certain embodiments, methods of treating a disease or disorder in asubject by in vivo genome editing of a cell of the subject by directlyadministering an AAV Clade F vector or an AAV vector variant to thesubject are disclosed. In certain embodiments, the AAV Clade F vector orAAV vector variant may comprise one or more Clade F capsids or capsidvariants, an editing element (targeting cassette) comprising one or moretherapeutic nucleotide sequences to be integrated into a target locus(target site) of the genome, a 5′ homologous arm polynucleotide sequenceflanking the editing element (targeting cassette) and having homology toa region that is upstream of the target locus (target site), and a 3′homologous arm polynucleotide sequence flanking the editing element(targeting cassette) and having homology to a region that is downstreamof the target locus (target site), wherein the vector transduces a cellof the subject and integrates the one or more therapeutic nucleotidesequences into the genome of the cell. In certain embodiments, the oneor more Clade F capsids or capsid variants may comprise a polypeptidesequence selected from the group of AAVF1 (SEQ ID NO: 2), AAVF2 (SEQ IDNO: 3), AAVF11 (SEQ ID NO: 4), AAVF3 (SEQ ID NO: 5), AAVF4 (SEQ ID NO:6), AAVF6 (SEQ ID NO: 7), AAVF7 (SEQ ID NO: 8), AAVF8 (SEQ ID NO: 9),AAVF9 (SEQ ID NO: 10), AAVF5 (SEQ ID NO: 11), AAVF12 (SEQ ID NO: 12),AAVF17 (SEQ ID NO: 13), AAVF13 (SEQ ID NO: 14), AAVF14 (SEQ ID NO: 15),AAVF15 (SEQ ID NO: 16), AAVF16 (SEQ ID NO: 17), variants, fragments,mutants, and any combination thereof. In certain embodiments, the one ormore Clade F capsids or capsid variants may comprise a polypeptidesequence of AAVF7 (SEQ ID NO: 8) or AAVF17 (SEQ ID NO: 13). In certainembodiments, the one or more Clade F capsids or capsid variants maycomprise a polypeptide sequence of AAVF5 (SEQ ID NO: 11), AAVF (SEQ IDNO: 8) or AAVF17 (SEQ ID NO: 13). In certain embodiments, the AAV CladeF vector or AAV vector variant does not contain a promoter for the oneor more therapeutic nucleotide sequences. In certain embodiments, thetarget locus (target site) may be a safe harbor site. In certainembodiments, the safe harbor site may be the AAVS1 locus on chromosome19. In certain embodiments, the cell may be a stem cell. In certainembodiments, the stem cell may be a hematopoietic stem cell, apluripotent stem cell, an embryonic stem cell, or a mesenchymal stemcell. In certain embodiments, the disease or disorder may be caused byone or more mutations in the cell genome. In certain embodiments, thedisease or disorder may be selected from an inherited metabolic disease,lysosomal storage disease, mucopolysaccharidodosis, immunodeficiencydisease, and hemoglobinopathy disease and infection.

Further demonstrating the efficacy of vivo applications, transplantationof transduced cells to immune-deficient mice with the isolate variants(relative to AAV9) resulted in prolonged and sustained transgeneexpression and may be used for gene therapy. In certain embodiments,when delivered systemically, these vectors display a tropism for theliver and cartilage, with implications for therapy of inherited,acquired, infectious and oncologic diseases. With respect to the livertransduction, the present AAV isolates have up to approximately 10-foldhigher liver transduction levels than the current gold standard forsystemic gene delivery to the liver, AAV8. This property can beexploited for gene-based enzyme replacement therapy from the liver fordiseases such as hemophilia, enzyme deficiency diseases, andatherosclerosis. The additional tropism of the present AAV isolates forcartilaginous tissue in joints may be exploited for the treatment ofbone disorders such as arthritis, osteoporosis or other cartilage/bonebased diseases. The variant sequences and methods may accordingly beused for transient transduction where long term integration is notdesirable.

Members of the AAV Clade F capsid family or AAV capsid variant familytransduce HSC, e.g. AAVF 15 and AAVF 17, giving rise to long-termengraftment with sustained gene expression and are thus strongcandidates for stem cell gene therapy vectors. AAVF17 and AAVF15 (alsoreferred in abbreviated form as “HSC17” and “HSC15”) supported thehighest levels of long-term in vivo transduction, up to 22 weekspost-transplantation. Serial bioluminescent imaging followingintravenous injection of the AAV variants revealed that AAVF15 generallysupported the highest levels of long-term transgene expression in vivo.Other AAV variants including AAVF13 and 17 also supported strong in vivotransduction.

AAVF15 was found to be highly liver tropic, about 5-10 fold higher thanAAV9. AAVF13 and AAVF15 also transduced the heart and skeletal muscle atleast 10-fold better than AAV9. In vitro neutralization titers revealedthat the prevalence of antibodies to AAVF 1-9 capsids in pooled humanIVIG were similar to AAV9, while antibodies to AAVF13, AAVF15, AAVF16and AAVF17 were somewhat less prevalent. In vivo neutralization assaysconfirmed that over 100-fold higher vector genome copies/cell were foundin liver and muscle following IVIG administration with AAVF15 comparedto AAV9, suggesting that pre-existing antibodies did not completelyneutralize AAVF15. Muscle diseases or disorders may comprise any cell,tissue, organ, or system containing muscle cells which have a disease ordisorder, including the heart, such as coronary heart disease orcardiomyopathy.

In addition, site-specific mutagenesis experiments indicate that theR505G mutation in AAVF15 is responsible for the enhanced liver tropism.The AAV Clade F vectors or AAV vector variants may be used to treat awhole host of genetic diseases such as hemophilia, atherosclerosis and avariety of inborn errors of metabolism. In one instance, AAVF15effectively treats hemophilia B. Some members of this family also targetthe joints after systemic injection, which may be used to treat jointand cartilage diseases such as arthritis. Other members of the familytarget the heart upon intravenous injection. Yet other members of thefamily target the brain. In some embodiments, a vector comprising AAVF5capsid proteins is provided as part of a method, kit or compositionprovided herein, as AAVF5 was shown to transduce multiple cell types(see FIG. 4 ).

In certain embodiments, methods of treating a neurological disease ordisorder in a subject by genome editing may comprise administering anAAV Clade F vector or AAV vector variant capable of crossing theblood-brain barrier, blood-ocular barrier, or blood-nerve barrier.Certain of the AAV Clade F vectors or AAV vector variants disclosedherein have the unique ability to traverse the biological junctions thatwere previously unknown to be accessible to any vector for gene therapyor other diagnostic or therapeutic purposes using a modified viralvector. These junctions have common characteristics. The blood-brainbarrier is a separation between blood circulating in the body and thebrain extracellular fluid in the central nervous system and is createdby tight junctions around capillaries. The blood-brain barrier generallyallows only the passage of by diffusion of small hydrophobic molecules.The blood-ocular barrier is a separation made by between the local bloodvessels and most parts of the eye and is made by endothelium ofcapillaries of the retina and iris. The blood-nerve barrier is thephysiological space within which the axons, Schwann cells, and otherassociated cells of a peripheral nerve function and is made ofendoneurial microvessels within the nerve fascicle and the investingperineurium. As with three of these barriers, there is restrictedpermeability to protect in the internal environment, here, the nerve,from drastic concentration changes in the vascular and otherextracellular spaces. The vector that traverses any of these barriershas a unique ability to deliver one or more therapeutic nucleotidesequences for treating the neurological disease or disorder or to act asa labeled and or diagnostic agent. Certain of the AAV Clade F vectors orAAV vector variants that have been experimentally validated as beingparticularly well suited for crossing these biological barriers includeAAVF15, AAVF15 A346T, and AAVF15 R505G.

There are many neurological diseases or disorders that are well known toone of skill in the art, which may be generally classified by cell ororgan-type such as a disease or disorder of the brain, spinal cord,ganglia, motor nerve, sensory nerve, autonomic nerve, optic nerve,retinal nerve, and auditory nerve. By way of example, brain diseases ordisorders may include cancer or other brain tumor, inflammation,bacterial infections, viral infections, including rabies, amoeba orparasite infections, stroke, paralysis, neurodegenerative disorders suchas Alzheimer's Disease, Parkinson's Disease, or other dementia orreduction in cognitive functioning, plaques, encephalopathy,Huntington's Disease, aneurysm, genetic or acquired malformations,acquired brain injury, Tourette Syndrome, narcolepsy, musculardystrophy, tremors, cerebral palsy, autism, Down Syndrome, attentiondeficit and attention deficit hyperactivity disorder, chronicinflammation, epilepsy, coma, meningitis, multiple sclerosis, myastheniagravis, various neuropathies, restless leg syndrome, and Tay-Sachsdisease.

Muscle diseases or disorders include, by way of example only,myopathies, chronic fatigue syndrome, fibromyalgia, muscular dystrophy,multiple sclerosis, atrophy, spasms, cramping, rigidity, variousinflammations, such as dermatomyositis, rhabdomyolysis, myofacial painsyndrome, swelling, compartment syndrome, eosinophilia-myalgia syndrome,mitochondrial myopathies, myotonic disorder, paralysis, tendinitis,polymyalgia rheumatic, cancer, and tendon disorders such as tendinitisand tenosynovitis.

Heart diseases or disorders include, by way of example only, coronaryartery disease, coronary heart disease, congestive heart failure,cardiomyopathy, myocarditis, pericardial disease, congenital heartdisease, cancer, endocarditis, and valve disease.

Lung diseases or disorders include, by way of example only, asthma,allergies, chronic obstructive pulmonary disease, bronchitis, emphysema,cystic fibrosis, pneumonia, tuberculosis, pulmonary edema, cancer, acuterespiratory distress syndrome, pneumonconiosis, and interstitial lungdisease.

Liver diseases or disorders include, by way of example only, cancer,hepatitis A, B, and C, cirrhosis, jaundice, and liver disease. Kidneydiseases or disorders include, by way of example only, cancer, diabetes,nephrotic syndrome, kidney stones, acquired kidney disease, congenitaldisease, polycystic kidney disease, nephritis, primary hyperoxaluria,and cystinuria. Spleen diseases or disorders include, by way of exampleonly, cancer, splenic infarction, sarcoidosis, and Gaucher's disease.Bone diseases or disorders include, by way of example only,osteoporosis, cancer, low bone density, Paget's disease, and infection.

With any of these diseases or disorders treated using therapeuticnucleotide sequences or even small molecules transported by or with theAAV Clade F vectors or AAV vector variants, the therapeutic nucleotidesequence may be, by way of example, a nucleic acid encoding a proteintherapeutic, such as for cancer—an apoptotic protein, miRNA, shRNA,siRNA, other RNA-subtypes or a combination thereof. In some embodiments,the vectors are isolated and purified as described herein. Isolation andpurification are preferred in vivo administration to increase efficacyand reduce contamination. The vector may permanently or transientlytransduce a transgene, which is a gene or other genetic material thathas been isolated from one organism and introduced into another. Here,the other organism may be the subject receiving the vector.

In certain embodiments, the AAV Clade F vectors or AAV vector variantsfor genome editing may be selected based on experimental results of thehighest efficacy in the given target cell or tissue for the givendisease or disorder as shown herein. For example a) for muscle diseaseor disorders and for antibody genes or other vaccine treatmentsadministered to the subject via the muscle, the AAV Clade F vector orAAV vector variant selected from the group of AAVF5, AAVF7, AAVF13,AAVF15, and AAVF17; b) for heart and lung disease or disorders, thevector selected from the group of AAVF13, AAVF15, and AAVF17; c) forliver or neurological diseases or disorders, the vector selected fromAAVF5 and AAVF15; d) for conditions treated by engrafting stem cells,vector AAVF17; e) for conditions treated by transducing B cellprogenitors, vector AAVF5; f) for conditions treated by transducingmyeloid and erythroid progenitors, vector AAVF12; and g) for lymph node,kidney, spleen, cartilage and bone disease or disorders, the vectorselected from the group of the vector selected from the group of AAVF7,AAVF13, AAVF15, and AAVF17; wherein the AAV Clade F vector or AAV vectorvariant transduces the cell or tissue and the one or more therapeuticnucleotide sequences are integrated into the genome of the cell andtreat the disease or disorder. In certain embodiments, the AAV Clade Fvector or AAV vector variant may comprise one or more Clade F capsids orcapsid variants (relative to AAV9) that demonstrates tropism for a cellas described herein.

The subject is any animal for which the method works, but is preferablya mammal, which may be a human. If the vector contains an antibody geneor other vaccine treatment it may be administered via injection in themuscle and may provide immunological protection against diseasesincluding from HIV, influenza, malaria, tetanus, measles, mumps,rubella, HPV, pertussis, or any other vaccine. The vector may bepackaged, isolated, and purified and may transduce a stem cell of anytype with the at least one therapeutic nucleotide sequence. The vectormay also transduce a transgene or carry corrective genes endogenous tothe subject and/or to the other subjects of the same species.

“AAV” is an adeno-associated virus. The term may be used to refer to thevirus or derivatives thereof, virus subtypes, and naturally occurringand recombinant forms, unless otherwise indicated. AAV has over 100different subtypes, which are referred to as AAV-1, AAV-2, etc., andincludes both human and non-human derived AAV. There are about a dozenAAV serotypes. The various subtypes of AAVs can be used as recombinantgene transfer viruses to transduce many different cell types.

“Recombinant,” as applied to a polynucleotide means that thepolynucleotide is the product of various combinations of cloning,restriction or ligation steps, and other procedures that result in aconstruct that is distinct from a naturally-occurring polynucleotide. Arecombinant virus is a viral particle comprising a recombinantpolynucleotide, including replicates of the original polynucleotideconstruct and progeny of the original virus construct. A “rAAV vector”refers to a recombinant AAV vector comprising a polynucleotide sequencenot of AAV origin (i.e., a polynucleotide heterologous to AAV), which isusually a sequence of interest for the genetic transformation of a cell.

A “helper virus” for AAV as used herein is virus that allows AAV to bereplicated and packaged by a mammalian cell. Helper viruses for AAV areknown in the art, and include, for example, adenoviruses (such asAdenovirus type 5 of subgroup C), herpes viruses (such as herpes simplexviruses, Epstein-Bar viruses, and cytomegaloviruses) and poxviruses.

“Joint tissue” is comprised of a number of tissues including cartilage,synovial fluid, and mature, progenitor and stem cells that give rise to,or are: (i) cartilage producing cells; (ii) Type I synoviocytes; (iii)Type II synoviocytes; (iv) resident or circulating leukocytes; (v)fibroblasts; (vi) vascular endothelial cells; and (vii) pericytes.

A “replication-competent” virus refers to a virus that is infectious andcapable of being replicated in an infected cell. In the case of AAV,replication competence generally requires the presence of functional AAVpackaging genes, as well as helper virus genes, such as adenovirus andherpes simplex virus. In general, rAAV vectors arereplication-incompetent (also referred to herein asreplication-defective) because they lack of one or more AAV packaginggenes. In some embodiments, an AAV may be considered replicationdefective (or replication-incompetent) if the AAV has an essentialabsence of an AAV rep gene and/or an AAV cap gene. In some embodiments,an AAV may be considered replication defective (orreplication-incompetent) if the AAV lacks an AAV rep gene and/or an AAVcap gene. In some embodiments, a composition comprising AAV Clade Fvectors or AAV variant isolates is a cell-free composition. Thecomposition is generally free of cellular proteins and/or othercontaminants and may comprise additional elements such as a buffer(e.g., a phosphate buffer, a Tris buffer), a salt (e.g., NaCl, MgCl2),ions (e.g., magnesium ions, manganese ions, zinc ions), a preservative,a solubilizing agent, or a detergent, (e.g., a non-ionic detergent;dimethylsulfoxide).

In another embodiment, an expression cassette comprises a polynucleotidesequence encoding a polypeptide comprising one or more of the Clade Fcapsids or AAV variant isolates, wherein the polynucleotide sequenceencoding the polypeptide comprises a sequence having at least about 95%,96%, 97%, more preferably about 98%, and most preferably about 99%sequence identity to the sequences taught in the present specification.Percentage identity may be calculated using any of a number of sequencecomparison programs or methods such as the Pearson & Lipman, Proc. Natl.Acad. Sci. USA, 85:2444 (1988), and programs implementing comparisonalgorithms such as GAP, BESTFIT, FASTA, or TFASTA (from the WisconsinGenetics Software Package, Genetics Computer Group, 575 Science Drive,Madison, Wis.), or BLAST, available through the National Center forBiotechnology Information web site.

In another aspect, an expression cassette comprises a polynucleotidesequence encoding a polypeptide comprising one or more of the Clade Fcapsids or AAV variant isolates, wherein the sequence is comprised ofportions of the three genes comprising the capsid protein, V1-V3 (alsoreferred to as VP1-VP3). For example, the cassette may comprise V1 fromcapsid AAVF1, a standard V2 as compared to AAV9 hu.14, and V3 fromAAVF17 capsids. In yet another embodiment, a capsid may comprise morethan one of each of the capsid gene components. For example, Clade Fcapsids or capsid variants may be selected from any of the VP1-VP3(V1-V3) for the capsid sequences set forth herein and may be combined inany order and in any combination so long as the desired property ofincreased transduction is achieved. For example, the capsid sequencecould be VP1A-VP1B-VP2-VP3 (V1A-V1B-V2-V3), VP3-VP1-VP2 (V3-V1-V2), orVP1-VP2-VP3A-VP3B (V1-V2-V3A-V3B).

Another embodiment includes methods of immunization of a subject.Compositions comprising the Clade F capsids or capsid variants may beintroduced into a subject in a manner that causes an immunologicalreaction resulting in immunity in the subject. The Clade F capsids orcapsid variants may be in the composition alone or as part of anexpression cassette. In one embodiment, the expression cassettes (orpolynucleotides) can be introduced using a gene delivery vector. Thegene delivery vector can, for example, be a non-viral vector or a viralvector. Exemplary viral vectors include, but are not limited toSindbis-virus derived vectors, retroviral vectors, and lentiviralvectors. Compositions useful for generating an immunological responsecan also be delivered using a particulate carrier. Further, suchcompositions can be coated on, for example, gold or tungsten particlesand the coated particles delivered to the subject using, for example, agene gun. The compositions can also be formulated as liposomes. In oneembodiment of this method, the subject is a mammal and can, for example,be a human.

The term “affinity tag” is used herein to denote a polypeptide segmentthat can be attached to a second polypeptide to provide for purificationor detection of the second polypeptide or provide sites for attachmentof the second polypeptide to a substrate. In principal, any peptide orprotein for which an antibody or other specific binding agent isavailable can be used as an affinity tag. Affinity tags include apoly-histidine tract, protein A (Nilsson et al., EMBO J. 4:1075, 1985;Nilsson et al., Methods Enzymol. 198:3, 1991), glutathione S transferase(Smith and Johnson, Gene 67:31, 1988), Glu-Glu affinity tag(Grussenmeyer et al., Proc. Natl. Acad. Sci. USA 82:7952-4, 1985),substance P, Flag™ peptide (Hopp et al., Biotechnology 6:1204-10, 1988),streptavidin binding peptide, or other antigenic epitope or bindingdomain. See, in general, Ford et al., Protein Expression andPurification 2: 95-107, 1991, DNAs encoding affinity tags are availablefrom commercial suppliers (e.g., Pharmacia Biotech, Piscataway, N.J.).

Of the number of affinity tag purification systems available, the mostfrequently employed utilize polyhistidine (His) or glutathioneS-transferase (GST) tags. His binds with good selectivity to matricesincorporating Ni+2 ions, typically immobilized with either iminodiaceticacid or nitrilotriacetic acid chelating groups. The technique is knownas immobilized metal affinity chromatography. Absorption of theHis-tagged protein is performed at neutral to slightly alkaline pH toprevent protonation and loss of binding capacity of the weakly basichistidine imidazole groups. Elution of the bound protein is caused bydisplacement with imidazole or low pH conditions.

Methods of generating induced pluripotent stem cells from somatic cellswithout permanent introduction of foreign DNA are also described. Themethod involved transiently transducing stem cells with vectorscomprising a Clade F capsid or capsid variant nucleotide sequence asdescribed herein encoding a polypeptide sequence, or VP1 (V1) or VP3(V3) portion thereof.

For these and other experiments, a person skilled in the art knows howto modify and propagate AAV. For example, AAV-2 can be propagated bothas lytic virus and as a provirus. For lytic growth, AAV requiresco-infection with a helper virus. Either adenovirus or herpes simplexcan supply helper function. When no helper is available, AAV can persistas an integrated provirus, which involves recombination between AAVtermini and host sequences and most of the AAV sequences remain intactin the provirus. The ability of AAV to integrate into host DNA allowspropagation absent a helper virus. When cells carrying an AAV provirusare subsequently infected with a helper, the integrated AAV genome isrescued and a productive lytic cycle occurs. The construction of rAAVvectors carrying particular modifications and the production of rAAVparticles, e.g., with modified capsids, is described, e.g., in Shi etal. (2001), Human Gene Therapy 12:1697-1711; Rabinowitz et al. (1999),Virology 265:274-285; Nicklin et al. (2001), Molecular Therapy4:174-181; Wu et al. (2000), J. Virology 74:8635-8647; and Grifman etal. (2001), Molecular Therapy 3:964-974.

Yet another aspect relates to a pharmaceutical composition containing anAAV Clade F vector or AAV vector variant or AAV particle as describedherein. The pharmaceutical composition containing an AAV Clade F vectoror AAV vector variant or particle, preferably, contains apharmaceutically acceptable excipient, adjuvant, diluent, vehicle orcarrier, or a combination thereof. A “pharmaceutically acceptablecarrier” includes any material which, when combined with an activeingredient of a composition, allows the ingredient to retain biologicalactivity and without causing disruptive physiological reactions, such asan unintended immune reaction. Pharmaceutically acceptable carriersinclude water, phosphate buffered saline, emulsions such as oil/wateremulsion, and wetting agents. Compositions comprising such carriers areformulated by well-known conventional methods such as those set forth inRemington's Pharmaceutical Sciences, current Ed., Mack Publishing Co.,Easton Pa. 18042, USA; A. Gennaro (2000) “Remington: The Science andPractice of Pharmacy”, 20th edition, Lippincott, Williams, & Wilkins;Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) H. C. Anselet al., 7th ed., Lippincott, Williams, & Wilkins; and Handbook ofPharmaceutical Excipients (2000) A. H. Kibbe et al., 3rd ed. Amer.Pharmaceutical Assoc. Such carriers can be formulated by conventionalmethods and can be administered to the subject at a suitable dose.Administration of the suitable compositions may be effected by differentways, e.g. by intravenous, intraperitoneal, subcutaneous, intramuscular,topical or intradermal administration. In some embodiments, thecomposition is formulated for administration to a mammal. In someembodiments, the composition is formulated for administration to amammal via intravenous injection, subcutaneous injection, intramuscularinjection, autologous cell transfer, or allogeneic cell transfer. Theroute of administration, of course, depends, inter alia, on the kind ofvector contained in the pharmaceutical composition. The dosage regimenwill be determined by the attending physician and other clinicalfactors. As is well known in the medical arts, dosages for any onepatient depends on many factors, including the patient's size, bodysurface area, age, sex, the particular compound to be administered, timeand route of administration, the kind and stage of infection or disease,general health and other drugs being administered concurrently.

Some of the AAV Clade F vectors or capsid variants are capable ofsupporting long-term stable transgene expression in vivo aftertransplantation of transduced hematopoietic stem cells or after directsystemic delivery of rAAV.

In certain embodiments, a nucleic acid comprising the Clade F capsids orAAV capsid isolate variants may be inserted into the genome of anewvirus, where in the addition of the Clade F capsid or capsid isolatevariant genes transmits the same or similar tissue or organ tropisms ofthe Clade F capsids or AAV capsid isolates to the new virus. Such genetherapy may be effected using in vivo and ex vivo gene therapyprocedures; see, e.g., U.S. Pat. No. 5,474,935; Okada, Gene Ther.3:957-964, 1996. Gene therapy using the AAV Clade F capsid or AAV capsidvariant gene will typically involve introducing the target gene in vitrointo the new virus, either alone or with another gene intended fortherapeutic purposes. If the tropic gene is introduced with one or moreadditional genes, preferably the resulting polypeptides are administeredfor therapeutic purposes in the tissue for which the Clade F capsid orAAV isolate has a tropism. The virus may then be administered to patientin need of such therapy or may be administered ex vivo, such as to anorgan awaiting transplant. The virus may be a retrovirus, an RNA virus,a DNA virus such as an adenovirus vector, an adeno-associated virusvector, a vaccinia virus vector, a herpes virus vector, and the like. Atransfection method using a virus vector that uses a liposome foradministration in which the new virus vector is encapsulated is alsocontemplated.

According to certain embodiments provided herein, kits are provided thatcomprise one or more AAV Clade F vectors or AAV vector variantsdescribed herein or compositions or formulations thereof. In certainembodiments, the one or more AAV Clade F vectors or AAV vector variantsin the kits may be used for genome editing of a cell. In certainembodiments, the kit may be used as a research tool to investigate theeffect of genome editing by the one or more AAV Clade F vectors or AAVvector variants.

Other aspects of the disclosure relate to a packaging system forrecombinant preparation of an AAV as described herein (e.g., an AAVClade F vector or an AAV variant vector) and methods of use thereof. Insome embodiments, the packaging system comprises a Rep nucleotidesequence encoding one or more AAV Rep proteins; a Cap nucleotidesequence encoding one or more AAV Cap proteins of an AAV Clade F capsidas described herein; and a correction genome as described herein,wherein the packaging system is operative in a cell for enclosing thecorrection genome in the capsid to form an adeno-associated virus.

In some embodiments, the packaging system comprises a first vectorcomprising the Rep nucleotide sequence and the Cap nucleotide sequence,and a second vector comprising the correction genome. As used in thecontext of a packaging system as described herein, a “vector” refers toa nucleic acid molecule that is a vehicle for introducing nucleic acidsinto a cell (e.g., a plasmid, a virus, a cosmid, an artificialchromosome, etc.).

In some embodiments of the packaging system, the AAV Clade F capsidcomprises at least one or at least two proteins selected from Clade FVP1, Clade F VP2 and Clade F VP3. In some embodiments of the packagingsystem, the AAV Clade F capsid comprises Clade F VP1, Clade F VP2 andClade F VP3 proteins. In some embodiments of the packaging system, theAAV Clade F capsid is selected from the group consisting of AAV9,AAVHSC1, AAVHSC2, AAVHSC3, AAVHSC4, AAVHSC5, AAVHSC6, AAVHSC7, AAVHSC8,AAVHSC9, AAVHSC10, AAVHSC11, AAVHSC12, AAVHSC13, AAVHSC14, AAVHSC15,AAVHSC16, AAVHSC17, AAVHU31, and AAVHU32.

In some embodiments of the packaging system, the Rep nucleotide sequenceencodes an AAV2 Rep protein. In some embodiments of the packagingsystem, the AAV2 Rep protein encoded is at least one of Rep 78/68 or Rep68/52. In some embodiments of the packaging system, the nucleotidesequence encoding the AAV2 Rep protein comprises a nucleotide sequencethat encodes a protein having a minimum percent sequence identity to theAAV2 Rep amino acid sequence of SEQ ID NO:40, wherein the minimumpercent sequence identity is at least 70% (e.g., at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 98%, at least99%, or 100%) across the length of the amino acid sequence of the AAV2Rep protein.

Exemplary AAV2 Rep amino acid sequence (SEQ ID NO:40)—mpgfyeivikvpsdldehlpgisdsfvnwvaekewelppdsdmdlnliegapltvaeklqrdfltewrrvskapealffvqfekgesyfhmhvlvettgvksmvlgrflsqirekliqriyrgieptlpnwfavtktrngagggnkvvdecyipnyllpktqpelqwawtnmeqylsaclnlterkrlvaqhlthvsqtgegnkenqnpnsdapvirsktsarymelvgwlvdkgitsekqwiqedqasyisfnaasnsrsgikaaldnagkimsltktapdylvgqqpvedissnriykilelngydpqyaasvflgwatkkfgkmtiwlfgpattgktniaeaiahtvpfygcvnwtnenfpfndcvdkmviwweegkmtakvvesakailggskvrvdqkckssaqidptpvivtsntnmcavidgnsttfehqqplqdrmfkfeltrrldhdfgkvtkqevkdffrwakdhvvevehefyvkkggakkrpapsdadisepkrvresvaqpstsdaeasinyadryqnkcsrhvgmnlmlfpcrqcermnqnsnicfthgqkdclecfpvsesqpvsvvkkayqklcyihhimgkvpdactacdlvnvdlddcifeq

In some embodiments of the packaging system, the packaging systemfurther comprises a third vector, e.g., a helper virus vector. The thirdvector may be an independent third vector, integral with the firstvector, or integral with the second vector. In some embodiments, thethird vector comprises genes encoding helper virus proteins.

In some embodiments of the packaging system, the helper virus isselected from the group consisting of adenovirus, herpes virus(including herpes simplex virus (HSV)), poxvirus (such as vacciniavirus), cytomegalovirus (CMV), and baculovirus. In some embodiments ofthe packaging system, where the helper virus is adenovirus, theadenovirus genome comprises one or more adenovirus RNA genes selectedfrom the group consisting of E1, E2, E4 and VA. In some embodiments ofthe packaging system, where the helper virus is HSV, the HSV genomecomprises one or more of HSV genes selected from the group consisting ofUL5/8/52, ICPO, ICP4, ICP22 and UL30/UL42.

In some embodiments of the packaging system, the first, second, and/orthird vector are contained within one or more transfecting plasmids. Insome embodiments, the first vector and the third vector are containedwithin a first transfecting plasmid. In some embodiments the secondvector and the third vector are contained within a second transfectingplasmid.

In some embodiments of the packaging system, the first, second, and/orthird vector are contained within one or more recombinant helperviruses. In some embodiments, the first vector and the third vector arecontained within a recombinant helper virus. In some embodiments, thesecond vector and the third vector are contained within a recombinanthelper virus.

In some aspects, the disclosure provides a method for recombinantpreparation of an AAV as described herein (e.g., an AAV Clade F vectoror AAV variant vector), wherein the method comprises transfecting ortransducing a cell with a packaging system as described under conditionsoperative for enclosing the correction genome in the capsid to form theAAV as described herein (e.g., an AAV Clade F vector or AAV variantvector). Exemplary methods for recombinant preparation of an AAV includetransient transfection (e.g., with one or more transfection plasmidscontaining a first, and a second, and optionally a third vector asdescribed herein), viral infection (e.g. with one or more recombinanthelper viruses, such as a adenovirus, poxvirus (such as vaccinia virus),herpes virus (including herpes simplex virus (HSV), cytomegalovirus, orbaculovirus, containing a first, and a second, and optionally a thirdvector as described herein), and stable producer cell line transfectionor infection (e.g., with a stable producer cell, such as a mammalian orinsect cell, containing a Rep nucleotide sequence encoding one or moreAAV Rep proteins and/or a Cap nucleotide sequence encoding one or moreAAV Cap proteins of an AAV Clade F capsid as described herein, and witha correction genome as described herein being delivered in the form of atransfecting plasmid or a recombinant helper virus).

Other exemplary, non-limiting embodiments of the disclosure are providedbelow.

Embodiment 1. An adeno-associated virus (AAV) vector variant for editingthe genome of a stem cell comprising one or more capsid variants; atargeting cassette comprising one or more therapeutic nucleotidesequences to be integrated into a target site of the genome; a 5′homologous arm polynucleotide sequence flanking the targeting cassetteand having homology to a region that is upstream of the target site; anda 3′ homologous arm polynucleotide sequence flanking the targetingcassette and having homology to a region that is downstream of thetarget site.

Embodiment 2. The AAV vector variant of embodiment 1, wherein the one ormore capsid variants comprise a polypeptide sequence selected from thegroup of HSC7 (SEQ ID NO: 8), HSC12 (SEQ ID NO: 12), HSC15 (SEQ ID NO:16), HSC17 (SEQ ID NO: 13), variants, fragments, mutants and anycombination thereof.

Embodiment 3. The AAV vector variant of embodiment 2, wherein the one ormore capsid variants comprise a polypeptide sequence having a percentsequence identity of at least 95% to a polypeptide sequence selectedfrom the group of HSC7 (SEQ ID NO: 8), HSC12 (SEQ ID NO: 12), HSC15 (SEQID NO: 16), HSC17 (SEQ ID NO: 13), variants, fragments, mutants and anycombination thereof.

Embodiment 4. The AAV vector variant of embodiment 1, wherein the targetsite is a safe harbor site.

Embodiment 5. The AAV vector variant of embodiment 4, wherein the safeharbor site is the AAVS1 locus on chromosome 19.

Embodiment 6. The AAV vector variant of embodiment 1, wherein the stemcell is a hematopoietic stem cell, a pluripotent stem cell, an embryonicstem cell, or a mesenchymal stem cell.

Embodiment 7. A method of editing the genome of a stem cell, comprisingtransducing, without additional exogenous nucleases, the stem cell withone or more adeno-associated virus (AAV) vector variants comprising oneor more capsid variants; a targeting cassette comprising one or moretherapeutic nucleotide sequences to be integrated into a target site ofthe genome; a 5′ homologous arm polynucleotide sequence flanking thetargeting cassette and having homology to a region that is upstream ofthe target site; and a 3′ homologous arm polynucleotide sequenceflanking the targeting cassette and having homology to a region that isdownstream of the target site.

Embodiment 8. The method of embodiment 7, wherein the one or more capsidvariants comprise a polypeptide sequence selected from the group of HSC7(SEQ ID NO: 8), HSC12 (SEQ ID NO: 12), HSC15 (SEQ ID NO: 16), HSC17 (SEQID NO: 13), variants, fragments, mutants and any combination thereof.

Embodiment 9. The method of embodiment 7, wherein the AAV vector variantdoes not contain a promoter for the one or more therapeutic nucleotidesequences.

Embodiment 10. The method of embodiment 7, wherein the target site is asafe harbor site.

Embodiment 11. The method of embodiment 10, wherein the safe harbor siteis the AAVS1 locus on chromosome 19.

Embodiment 12. The method of embodiment 7, wherein the stem cell is ahematopoietic stem cell, a pluripotent stem cell, an embryonic stemcell, or a mesenchymal stem cell.

Embodiment 13. A method of treating a disease or disorder in a subjectby editing a genome of a stem cell of the subject, comprisingtransducing, without additional exogenous nucleases, the stem cell ofthe subject with an adeno-associated virus (AAV) vector variantcomprising one or more capsid variants; a targeting cassette comprisingone or more therapeutic nucleotide sequences to be integrated into atarget site in the genome of the stem cell; a 5′ homologous armpolynucleotide sequence flanking the targeting cassette and havinghomology to a region that is upstream of the target site; a 3′homologous arm polynucleotide sequence flanking the targeting cassetteand having homology to a region that is downstream of the target site;and transplanting the transduced stem cell into the subject, wherein thetransduced stem cell treats the disease or disorder.

Embodiment 14. The method of embodiment 13, wherein the one or morecapsid variants comprise a polypeptide sequence from the group of HSC7(SEQ ID NO: 8), HSC12 (SEQ ID NO: 12), HSC15 (SEQ ID NO: 16), HSC17 (SEQID NO: 13), variants, fragments, mutants and any combination thereof.

Embodiment 15. The method of embodiment 13, wherein the AAV vectorvariant does not contain a promoter for the one or more therapeuticnucleotide sequences.

Embodiment 16. The method of embodiment 13, wherein the target site is asafe harbor site.

Embodiment 17. The method of embodiment 16, wherein the safe harbor siteis the AAVS1 locus on chromosome 19.

Embodiment 18. The method of embodiment 13, wherein the stem cell is ahematopoietic stem cell, a pluripotent stem cell, an embryonic stemcell, or a mesenchymal stem cell.

Embodiment 19. The method of embodiment 13, wherein the disease ordisorder is caused by one or more mutations in the cell genome.

Embodiment 20. The method of embodiment 19, wherein the disease ordisorder is selected from an inherited metabolic disease, lysosomalstorage disease, mucopolysaccharidodosis, immunodeficiency disease, andhemoglobinopathy disease and infection.

Embodiment 21. A method of treating a disease or disorder in a subjectby in vivo genome editing of a cell of the subject by directlyadministering an AAV vector variant to the subject, said vectorcomprising one or more capsid variants; a targeting cassette comprisingone or more therapeutic nucleotide sequences to be integrated into atarget site of the genome; a 5′ homologous arm polynucleotide sequenceflanking the targeting cassette and having homology to a region that isupstream of the target site; and a 3′ homologous arm polynucleotidesequence flanking the targeting cassette and having homology to a regionthat is downstream of the target site, wherein the vector transduces thecell of the subject and integrates the one or more therapeuticnucleotide sequences into the genome of the cell.

Embodiment 22. The method of embodiment 21, wherein the one or morecapsid variants comprise a polypeptide sequence selected from the groupof HSC1 (SEQ ID NO: 2), HSC2 (SEQ ID NO: 3), HSC11 (SEQ ID NO: 4), HSC3(SEQ ID NO: 5), HSC4 (SEQ ID NO: 6), HSC6 (SEQ ID NO: 7), HSC7 (SEQ IDNO: 8), HSC8 (SEQ ID NO: 9), HSC9 (SEQ ID NO: 10), HSC5 (SEQ ID NO: 11),HSC12 (SEQ ID NO: 12), HSC17 (SEQ ID NO: 13), HSC13 (SEQ ID NO: 14),HSC14 (SEQ ID NO: 15), HSC15 (SEQ ID NO: 16), HSC16 (SEQ ID NO: 17),variants, fragments, mutants, and any combination thereof.

Embodiment 23. The method of embodiment 21, wherein the cell is a stemcell.

Embodiment 24. The method of embodiment 23, wherein the stem cell is ahematopoietic stem cell, a pluripotent stem cell, an embryonic stemcell, or a mesenchymal stem cell.

Embodiment 25. The method of embodiment 24, wherein the disease ordisorder is caused by one or more mutations in the cell genome.

Embodiment 26. The method of embodiment 25, wherein the disease ordisorder is selected from an inherited metabolic disease, lysosomalstorage disease, mucopolysaccharidodosis, immunodeficiency disease, andhemoglobinopathy disease and infection.

Embodiment 27. The method of embodiment 21, wherein the AAV vectorvariant does not contain a promoter for the one or more therapeuticnucleotide sequences.

Embodiment 28. The method of embodiment 22, wherein the target site is asafe harbor site.

Embodiment 29. The method of embodiment 23, wherein the safe harbor siteis the AAVS1 locus on chromosome 19.

The following examples are intended to illustrate various embodiments ofthe disclosure. As such, the specific embodiments discussed are not tobe construed as limitations on the scope of the invention. It will beapparent to one skilled in the art that various equivalents, changes,and modifications may be made without departing from the scope of thedisclosure, and it is understood that such equivalent embodiments are tobe included herein. Further, all references cited in the disclosure arehereby incorporated by reference in their entirety, as if fully setforth herein.

EXAMPLES Example 1: AAV Clade F Vector Variant Mediated Genome Editingin a CD34+ Human Hematopoietic Stem Cell Line or a K562 Cell Line

Genome editing through site-specific insertion or targeted integrationof specific DNA sequences using AAVF (AAVHSC) vectors without the use ofan exogenous nuclease was performed in human CD34+ hematopoietic celllines from healthy donors or the K562 cell line, which is a CD34+erythroleukemic cell line. One set of donor recombinant AAV vectors,ITR-AAVS1-FP vectors, was constructed and was used to integrateatransgene into the AAVS1 locus on chromosome 19, the natural wild-typeAAV integration site (Kotin, 1992; Giraud, 1994). The AAVS1 locus onchromosome 19 qter13.3-13.4 was previously shown to be a “safe harbor”site for the insertion of transgenes since genes inserted here areexpressed with no pathogenic consequences, which is similar to wild-typeAAV that integrates at this locus with no pathogenic consequences(Giraud, 1994; Linden, 1996A; Linden 1996B). The transgene to beintegrated was a Venus yellow fluorescent protein (“YFP” or “FP”) gene,which was flanked on each side by approximately 800 nucleotides havinghomology with the AAVS1 locus on human chromosome 19 (see schematic inFIG. 3 ). The donor AAV vector was designed such that the transgene waspromoterless and would only be expressed if it was integrated at thecorrect locus, which would be downstream from chromosomally encodedregulatory sequences (see FIG. 4 ). Thus, any Venus YFP transgeneexpression that occurred was under the control of a chromosomal promoterlocated in or near AAVS1.

The donor vector, ITR-AAVS1-FP, was packaged into AAVHSC capsidsaccording to the standard AAV packaging method described in Chatterjeeet al, 1992. Specifically, ITR-AAVS1-FP was packaged into AAVHSC7,AAVHSC15, or AAVHSC17 capsids forming the pseudotyped AAVHSC-AAVS1-FPvector (i.e., a AAV vector variant). Human CD34+ hematopoietic stem celllines or K562 cells were transduced with the pseudotyped AAVHSC-AAVS1-FPat different multiplicities of infection (MOI) (i.e., 50,000 MOI;100,000 MOI; 200,000 MOI; 300,000 MOI; and 400,000 MOI).

Integration of the YFP transgene into the AAVS1 locus by homologousrecombination was initially assayed by cytofluorometric analysis of YFPexpression in transduced K562 cells. Targeted integration using theAAVHSC7 FP vector resulted in expression of the YFP transgene 24 hourspost-transduction (FIGS. 5 and 7A) and 72 hours post-transduction (FIGS.6 and 7B). Additionally, as the MOI of the AAVHSC7 FP vector wasincreased, the average percentage of YFP expression also increased(FIGS. 7A and B).

Targeted integration of the YFP transgene was further confirmed by PCRamplification of the edited genome using primers located outside of thehomology regions. Briefly, DNA was extracted from K562 cells transducedat an MOI of 100,000 vector genomes/cell with AAVHSC7 FP vector. PCRamplification was performed using the “OUT Forward Primer Region” and“OUT Reverse Primer Region” primers (see FIG. 4 ). Integration of theYFP transgene resulted in an increase in size of the AAVS1 locus fromthe wild type size of ˜1.9 kb to the YFP transgene containing ˜3.1 kbfragment (see FIG. 4 , compare line labeled “Fragment 1” with linelabeled “Fragment 2”). Amplification of the ˜3.1 kb fragment containingthe YFP transgene within the chromosome 19 AAVS1 locus indicated thatthe YFP transgene was effectively integrated into the AAVS1 locus incells transduced with the AAVHSC7 FP vector (see FIGS. 8A and 8B, lane4).

Example 2: AAV Vector Variant Mediated Genome Editing in Primary HumanCD34+ Peripheral Blood Stem Cells

Genome editing through site-specific insertion or targeted integrationof specific DNA sequences using AAVHSC vectors without the use of anexogenous nuclease was also performed in human CD34+ primary peripheralblood-derived human hematopoietic stem cells (PBSCs). Briefly, thevector, ITR-AAVS1-FP, was packaged in AAVHSC capsids including AAVHSC7,AAVHSC12, AAVHSC15, and AAVHSC17 (see Chatterjee, 1992 for the standardAAV packaging method). Primary CD34+ cells were transduced with thepseudotyped AAVHSC-AAVS1-FP vector (i.e., a AAV vector variant) at MOIsof 100,000 and 150,000.

Integration of the YFP transgene into the AAVS1 locus by homologousrecombination was assayed by cytofluorometric analysis of YFPexpression. Targeted integration using the AAVHSC7 FP and AAVHSC17 FPvectors in primary CD34+ cells resulted in expression of the YFPtransgene 1 day post-transduction at an MOI of 150,000 (FIG. 9 ), 4 dayspost-transduction at an MOI of 100,000 (FIG. 10 ), and 18 dayspost-transduction at an MOI of 100,000 (FIG. 11 ). The percentage ofpositive cells expressing YFP at 5.5 weeks post transduction at an MOIof 100,000 (39 days) did not decline (see FIGS. 12A and B, compare 20day results with 39 day results). This long term expression of apromoterless YFP transgene in a dividing cell population indicatesaccurate integration of the transgene.

Targeted integration of the YFP transgene was further confirmed by PCRanalysis. The edited genome was amplified using primers that amplify the5′ junction region between the inserted transgene sequence and thenative chromosomal 5′ homology arm sequence (see FIG. 4 , see linelabeled “Fragment 3”). Briefly, DNA was extracted from primary CD34+cells transduced at an MOI of 150,000 vector genomes/cell with theAAVHSC7 FP vector. PCR amplification was performed using the “OUTForward Primer Region” and the “In Reverse Primer” primers (see FIG. 4). Amplification of a ˜1.7 kb fragment of the 5′ junction region fortransduced primary CD34+ cells indicated that the YFP transgene wassuccessfully integrated into the AAVS1 locus (see FIG. 13 , lane 5).Whereas, there was no amplified product for those cells that were nottransduced with the AAVHSC7 FP vector (see FIG. 13 , lane 3).

Targeted integration of the YFP transgene was further confirmed bysequence analysis of different portions of the edited AAVS1 locus.Sequencing was performed beginning near the “OUT Forward Primer Region”(see FIG. 14 ), near the 5′ homology arm (see FIG. 15 ), near the 5′region of the regulatory elements (see FIG. 16 ), near the 3′ region ofthe regulatory elements (see FIG. 17 ), near the 5′ region of thetransgene (see FIG. 18 ), and near the “IN Reverse Primer” region (seeFIG. 19 ). Sequencing results indicated that the YFP gene was presentand was successfully integrated into the AAVS1 locus.

As provided in Examples 1 and 2, the AAVHSC vectors successfullymediated efficient targeted genome editing in human CD34+ hematopoieticcell lines and CD34+ PBSCs without the need for addition of exogenousendonucleases. AAVHSC vectors were capable of directing integration ofthe YFP transgene to the AAVS1 locus on chromosome 19 based uponflanking homology arms corresponding to the AAVS1 locus. AAV-mediatedtransgenesis has previously been reported; however, reported frequencieshave been low, usually on the order of 1 in 1e6 cells to 1 in 1e4 cells.As shown herein, targeted genome editing using AAVHSC vectors occurredat frequencies of approximately 10% of primary cells long term, which is1,000 to 100,000 fold more efficient than previously reported (see,e.g., Khan, 2011). Expression of the YFP transgene in human CD34+hematopoietic cell lines was observed as early as day onepost-transduction and was confirmed on day three. Expression of the YFPtransgene in PBSCs was observed starting from day one and continued longterm (up to almost 6 weeks), which was the latest time point analyzed.No overt toxicity was observed as a result of AAVHSC vectortransduction. Based upon the high frequency of insertion, ease of use,and lack of toxicity observed, therapies based upon targeted genomeediting using AAVHSC vectors is practical and feasible.

Example 3: In Vivo Genome Engineering with AAV Vector Variants

AAVHSC vectors encoding luciferase and AAVHSC vectors encoding Venuswere injected into adult immune-deficient mice previously xenograftedwith human cord blood CD34+ HSCs. As shown below, intravenous injectionof AAVHSC vectors resulted in transduction of human CD34+ hematopoieticstem and progenitor cells in vivo, and the intravenous injected AAVHSCvectors trafficked to sites of human hematopoiesis and transduced humancells.

Methods. Briefly, immune-deficient NOD/SCID adult mice were firstirradiated with a sublethal dose of 350cGy from a ¹³⁷Cs source. Second,one million human cord blood CD34+ cells were injected into thesublethally-irradiated immune-deficient NOD/SCID mice. Next, two hoursafter CD34+ HSC transplantation, the mice were injected intravenouslywith approximately 1E11-5e11 particles of AAVHSC-Luciferase vector(either AAVHSC7-Luciferase vector or AAVHSC17-Luciferase vector). Thesevectors were used in the absence of an exogenous nuclease. TheAAVHSC-Luciferase vectors encode the single-stranded firefly luciferasegene (ssLuc) under the control of the ubiquitous CBA promoter to permitserial in vivo bioluminescent monitory of transgene expression. Thesevectors are described specifically in U.S. application Ser. No.13/668,120 (published as US Patent Publication Number 20130096182A1) andin Smith et al., which is hereby incorporated by reference in itsentirety, as if fully set forth herein (see Smith, 2014). TheAAVHSC-Luciferase vector was pseudotyped in the HSC7 capsid variant (thepolynucleotide sequence of HSC7 capsid is provided as SEQ ID NO: 27 andthe polypeptide sequence of HSC7 capsid is provided as SEQ ID NO:8 (seeFIG. 1 )) or the HSC17 capsid variant (the polynucleotide sequence ofHSC17 capsid is provided as SEQ ID NO: 35 and the polypeptide sequenceof HSC17 capsid is provided as SEQ ID NO:13 (see FIG. 1 )) as describedin U.S. application Ser. No. 13/668,120 (published as US PatentPublication Number 20130096182A1) and Smith et al, to form theAAVHSC7-Luciferase vector and the AAVHSC17-Luciferase vector (see Smith,2014) (see Chatterjee, 1992 for the standard AAV packaging method). Notethat the AAVHSC-Luciferase vectors can transduce both mouse and humancells. However, in contrast to the Venus encoded in the AAVHSC-Venusvector described below, the luciferase will not integrate into AAVS1.Instead, the luciferase may integrate randomly or stay episomal.

Two to seven days after injection with the AAVHSC-Luciferase vector, themice were injected with approximately 1E11-5e11 particles ofAAVHSC-Venus vectors. Specifically, mice that were first injected withAAVHSC7-Luciferace vector were injected with AAVHSC7-Venus vector andmice that were first injected with AAVHSC17-Luciferase vector wereinjected with AAVHSC17-Venus vector. The Venus donor vector used isdescribed specifically in Examples 1 and 2 above. The donor AAV vectorwas designed such that the Venus transgene was promoterless and wouldonly be expressed if it was integrated at the correct AAVS1 locus, whichwould be downstream from chromosomally encoded regulatory sequences (seeFIGS. 3 and 4 ). Thus, any Venus transgene expression that occurred wasunder the control of a chromosomal promoter located in or near AAVS1.Importantly, the vector containing the Venus gene does not contain apromoter to drive expression. The Venus gene will only be expressed ifit integrates correctly into AAVS1 in human cells, downstream from anendogenous chromosomal promoter. The donor vector, ITR-AAVS1-Venus (seeFIG. 3 ), was packaged into AAVHSC capsids according to the standard AAVpackaging method described in Chatterjee et al, 1992. The vector waspseudotyped in the HSC7 capsid variant (the polynucleotide sequence ofHSC7 capsid is provided as SEQ ID NO: 27 and the polypeptide sequence ofHSC7 capsid is provided as SEQ ID NO:8 (see FIG. 1 )) or the HSC17capsid variant (the polynucleotide sequence of HSC17 capsid is providedas SEQ ID NO: 35 and the polypeptide sequence of HSC17 capsid isprovided as SEQ ID NO:13 (see FIG. 1 )) to form the AAVHSC7-Venus vectorand the AAVHSC17-Venus vector.

Finally, in vivo luciferase expression was measured 4 weekspost-injection. Six weeks post-injection, engraftment of human CD34+ andCD45+ cells was measured and Venus expression was quantitated. See FIG.20 for an overall schematic of the experiments performed in thisExample.

Results. Four weeks post-injection, in vivo imaging was performed onxenotransplanted and non-xenotransplanted mice that received intravenousinjections of AAVHSC-Luciferase vectors. Results showed specificluciferase expression in vertebrae, spleen, hips, and long bones, whichare all sites of hematopoiesis after transplantation (see FIG. 21A).However, no specific luciferase expression in hematopoietic organs wasobserved in mice that were not previously xenografted with human cordblood CD34+ HSCs (see FIG. 21B). These results indicate thatintravenously injected AAVHSC vectors traffic to in vivo sites of humanhematopoiesis and preferentially transduce stem and progenitor cells.

Six weeks after injection with the AAVHSC-Venus vectors, human CD34+ andCD45+ cells were analyzed using flow cytometry. Results indicated thatthe injected human cord blood CD34+ cells engrafted into the mice andgave rise to more mature blood cells. Specifically, primitive humanblood progenitor cells (i.e., CD34+ cells) were observed in the bonemarrow (see Table 1, CD34+ cells and femoral marrow). Additionally,human mononuclear blood cells (i.e., CD45+ cells) were evident in thefemoral marrow, vertebral marrow, and spleen as shown in Table 1.

TABLE 1 Engraftment of Human Blood Cells in Immune Deficient Mice Celltype Femoral Marrow Vertebral Marrow Spleen AAVHSC7 CD45+ 68.4 30.4 24.3CD34+ 22 NT* NT AAVHSC17 CD45+ 46.6 24 18.3 CD34+ 13.1 NT NT *NT = NotTested CD45+ cells: human mononuclear blood cells CD34+ cells: humanhematopoietic progenitor cells

Six weeks post injection, flow cytometry was used to analyze Venusexpression from human HSCs of xenotransplanted mice that receivedintravenous injections of either AAVHSC7-Venus or AAVHSC17-Venusvectors. Results revealed that AAVHSC transduction was readily observedin the CD45+ human HSCs as well as CD34+ human HSCs from the femoral andvertebral marrow (see Table 2 and FIGS. 22A-F).

TABLE 2 Percentage of Engrafted Human Hematopoietic Cells ExpressingVenus Cell Type Femoral Marrow Vertebral Marrow Spleen AAVHSC7 CD45+8.35 15.3 10.3 CD34+ 9.23 NT* NT AAVHSC17 CD45+ 8.59 70.2 9.9 CD34+ 8.92NT NT *NT = Not Tested CD45+ cells: human mononuclear blood cells CD34+cells: human hematopoietic progenitor cells

Additionally, human CD45+ cells in the spleen readily showed evidence oftransduction as Venus was expressed in these cells (see Table 2 andFIGS. 22G and H). This demonstrates that CD45+ cells arising from thetransplanted human cord blood CD34+ cells express Venus. These resultsindicate that intravenous injection of AAVHSC vectors in vivo results intransduction of human hematopoietic cells.

Example 4: Insertion of Large and Small Editing Elements into a GenomeUsing AAV Clade F Vectors

Methods. rAAV Production, Purification, and Titration. All targetinggenomes were cloned into an AAV2 backbone using New England BiolabsGibson Assembly Cloning Kit with primers designed using NEBuilderv.1.6.2 (Ipswich, Mass.). All targeting genomes were sequenced and AAV2ITR integrity was confirmed using restrictions digest and sequencing.Single-stranded targeting genomes were packaged into the AAVF capsids inherpes simplex virus (HSV)-infected 293 cells. The resulting recombinantAAV vectors were purified through two rounds of CsCl2 densitycentrifugation gradients and titers were determined using qPCR withtransgene-specific primers and probe.

K562, HepG2 and PBSC Transductions. The chronic myelogenous leukemia(CML) cell line, K562 and the hepatocellular carcinoma cell line HepG2,were obtained from American Type Culture Collection (ATCC) (Manassas,Va.) and cultured according to ATCC guidelines. Peripheral blood stemcells (PBSCs) were purified from mononuclear cells from cytokine primedPB of healthy donors using CD34+ Indirect isolation kits (MiltenyiBiotech) and transductions performed immediately after isolation. PBSCswere cultured in Iscove's Modified Dulbecco's Medium (IMDM) (IrvineScientific) containing 20% FCS, 100 ug/mL streptomycin, 100 U/mLpenicillin, 2 mmol/L L-glutamine, IL-3 (10 ng/mL; R&D Systems), IL-6 (10ng/mL; R&D Systems), and stem cell factor (1 ng/mL; R&D Systems. HepG2cells were split and plated approximately 24 hours prior totransductions. K562 cells were plated immediately prior totransductions. K562s, HepG2s or PBSCs were transduced with AAVFtargeting vectors at MOIs ranging from 5E4 to 4E5. The cells weretransduced and homologous recombination was achieved in the absence ofan exogenous nuclease. Cells were harvested for flow and molecularanalysis at time points between 1 to 39 days post transduction. BrdUlabeling of in vitro transductions were performed prior to harvestingusing the APC BrdU Flow Kit (BD Biosciences) as instructed.

TI PCR and Sequencing. High molecular weight DNA was isolated fromK562s, HepG2s or PBSCs transduced with AAVF targeting vectors. TIspecific PCR was performed using a primer that anneal to the chromosomalregion outside the homology arms, Sigma AAVS1 forward primer (5′-GGC CCTGGC CAT TGT CAC TT-3′) and a primer that anneal to the insertedcassette, either Venus reverse primer (5′-AAC GAG AAG CGC GAT CAC A-3′)or RFLP HindIII reverse primer (5′-CCAATCCTGTCCCTAGTAAAGCTT-3′). RocheExpand Hifidelity PCR system (Indianapolis, Ind.) was used and cyclingconditions as follows: 1 cycle, 5 minutes—95° C.; 15 cycles, 30seconds—95° C., 30 seconds—start at 62° C. and decrease by 0.5° C. percycle, 2 minutes—68° C.; 20 cycles, 30 seconds—95° C., 30 seconds—53°C., 2 minutes—68° C.; 1 cycle, 5 minutes—68° C. PCR products were PCRpurified for direct sequencing using Qiaquick PCR Purification Kit(Qiagen) or cloned using TOPO TA Cloning Kit for Sequencing and clonessequenced by Sanger Sequencing (Life Technologies).

Transplantation of CD34+ Cells. All animal care and experiments wereperformed under protocols approved by a Institutional Animal Care andUse Committee. 6-8-week old male NOD.CB17-Prkdcscid/NCrCrl (NOD/SCID)mice were maintained in a specific pathogen free facility. Mice wereplaced on sulfamethoxazole and trimethoprim oral pediatric antibiotic(Hi-Tech Pharmacal (Amityville, N.Y.), 10 ml/500 ml H2O) for at least 48hours before transplant. Mice were irradiated with a sublethal dose of350cGy from a 137Cs source and allowed to recover for a minimum of 4hours prior to transplantation. Umbilical cord blood (CB) CD34+ cellswere isolated using CD34+ Indirect isolation kits (Miltenyi Biotech).1×10⁶ CB CD34+ cells were resuspended in approximately 200 ul andtransplanted by tail vein injection. 2.4E11 to 6.0E11 particles of AAVFtargeting vectors were injected intravenously through the tail vein at 1or 7 weeks post CB CD34+ transplantation. Femoral bone marrow (BM),vertebral BM and spleen were harvested 6, 7 or 19 week posttransplantation.

Flow Cytometric Analysis. In vitro transductions were analyzed for AAVFtargeting vector mediated integration, and BrdU and 7-AAD using a CyanADP Flow Cytometer (Dako). Specific fluorescence was quantifiedfollowing the subtraction of autofluorescence. In vivo expression ofintegrated fluorescent cassette in CD34+ and erthyroid cells wasanalyzed in harvested vertebral BM, femoral BM and spleen of xenograftedmice by staining with human specific antibodies, APC-conjugatedanti-CD34 and PE-conjugated anti-Glycophorin A, and PE- andAPC-conjugated IgG controls (BD Biosciences) on an FACS Aria SORP (BDBiosciences). Flow cytometry data was analyzed using FlowJo software(Treestar).

Results. Stem cell-derived AAV were shown to map to AAV clade F based onnucleotide sequence homology of the capsid genes (FIG. 23 , Smith et al,Mol Ther. 2014 September; 22(9):1625-34). These stem cell-derived AAVwere named AAVHSC1-17. These AAV are also referred to herein asAAVF1-17, respectively.

A singled stranded AAV vector genome was used to design a correctiongenome containing homology arms and a large insert (FIG. 24 ). Theinsert contained a promoterless Venus open reading frame (ORF)downstream from a splice acceptor (SA) and a 2A sequence (2A) to allowfor independent protein expression. Venus is a variant of yellowfluorescent protein (see, e.g., Nagai et al. Nat Biotechnol, 2002,20(1): 87-90). The left and right homology arms (HA) were each 800 bplong and were complementary to sequences in Intron 1 of the humanPPP1R12C located in the AAVS1 locus on chromosome 19 (FIG. 25 ). Asimilar single stranded AAV vector genome was designed with a 10 bpinsert between the two homology arms (FIG. 35 ). The AAVS1 locus isconsidered a safe harbor site for the insertion of heterologoustransgenes.

The homology arms, the open reading frame and regulatory sequences werecloned between AAV2 inverted terminal repeats (ITRs). This correctiongenome was then packaged (pseudotyped) in different AAV capsids,including AAVHSC, AAV8, 9, 6 and 2. Recombinant viruses were then usedto deliver the editing genome to the nuclei of target cells. Targetcells tested included CD34+ erythroleukemia cell lines, liver cell linesand primary human CD34+ hematopoietic stem/progenitor cells and as wellas their hematopoietic progeny.

AAVF vectors containing the Venus ORF, preceded by and flanked byhomology arms complementary to Intron 1 of the human PPP1R12C genes wereused to deliver the editing genome to primary human CD34+cytokine-primed peripheral blood stem cells (FIG. 26A), K562, a humanCD34+ erythroleukemia cell line (FIG. 26B), and HepG2, a human livercell line (FIG. 26C). Primary CD34+ cells supported the highest level ofediting, up to 60% (FIGS. 26A-F). Immortalized cell lines, includingK562 and HepG2, also showed significant levels of editing. In all cases,the level of editing achieved was consistently significantly higher thanthat achieved with non-Clade F viruses, including AAV6 and AAV8 (FIG.26A-F and FIG. 36 ).

In another experiment, DNA extracted from cytokine primed CD34+peripheral blood stem cells (PBSC) transduced with AAVF7, AAVF15 orAAVF17 vectors was amplified with a chromosome-specific primer and aninsert-specific primer. The vectors included either a large insert(Venus) or a short insert (10 bp, RFLP). Gels showed that correctlysized amplicons were amplified from the edited CD34 cells (FIG. 27 ,FIG. 35 , and FIG. 36 ). The presence of the 1.7 kb and 1 kb bandsreflected correctly targeted integration of large and small inserts,respectively. Targeted integration was shown at both short and long-termtime points after editing with AAVF vectors (FIG. 27 ).

In another experiment, single stranded AAV vector genomes were designedfor the insertion of a 10 bp insert in intron 1 of the human PPP1R12Cgene (FIG. 28A). These vectors included a wild type left homology arm(HA-L) which contained a Nhe1 restriction enzyme recognition site(GCTAGC). The NS mut vector, was designed to change the TA sequence inthe left homology arm on chromosome 19 to AT. This change results in theconversion of an Nhe1 site to an Sph1 site, changing the sequence fromGCTAGC to GCATGC. FIG. 28B shows the relative sizes of the expectedfragments created by cutting with Nhe1 or Sph1 when genomic DNA fromK562 cells was edited using either the wild type or the NS Mut AAVFvectors. Actual amplicons derived from genomic DNA of K562 cells editedwith a wild type AAVF vector were digested with Nhe 1, but not withSph1, as predicted (FIG. 28C). Results with amplified K562 DNA afterediting with AAVF7 or an AAVF17 vectors encoding either wild type or NSMut genomes showed that digestion with Nhe1 no longer resulted incleavage of the amplicon, comparable to the amplicon from uneditedcells. Digestion with Sph1 resulted in cleavage, demonstrating that theNhe1 site in the left homology arm of the chromosome was replaced by anSph1 site (FIG. 28D). Electrophoresis of amplified DNA form ahepatocellular carcinoma cell line, HepG2, after editing with AAVF7 oran AAVF17 vectors encoding either wild type or NS Mut genomes showedthat digestion with Nhe1 no longer resulted in cleavage of the amplicon,comparable to the amplicon from unedited cells (FIG. 28E). Digestionwith Sph1 resulted in cleavage, demonstrating that the Nhe1 site in theleft homology arm of the chromosome was replaced by an Sph1 site.Sequence analysis confirmed editing with AAVF7 and AAVF17 Wild type orNS Mut vectors (FIG. 29 ). These results demonstrate that both AAVF7 aswell as AAVF17 successfully mediated the 2 nucleotide substitution inthe chromosomal sequences in two different cell lines. These resultsalso demonstrate the ability of AAVF vectors to mediate a 2 base pairsubstitution in genomic DNA of human cells, suggesting their use forcorrection of disease-causing mutations or induction of new mutations inthe genome.

In another experiment, the potential requirement for cell division onthe editing capacity of AAVF vectors was tested on healthy human CD34+PBSC. BrdU was incorporated into transduced human CD34+ PBSC throughpulsing with 10 μM of BrdU for 2 hours. AAVF transduced CD34+ cells wereharvested, permeabilized and fixed prior to DNase treatment. After DNasetreatment, treated cells were stained with anti-BrdU APC antibody for 20minutes. BrdU labeling of in vitro edited cells was performed prior toharvesting using the APC BrdU Flow Kit (BD Biosciences) as perinstructions. Cells were then analyzed by flow cytometry for Venusexpression as well as BrdU labeling. Results revealed the similarfrequencies of Venus expression in both the BrdU positive and negativepopulations, suggesting that cell division was not required forAAVF-mediated editing (FIG. 30 ).

In another experiment, editing of engrafted human hematopoietic stemcells in vivo was tested by systemically delivered AAVF vectors immunedeficient NOD/SCID mice engrafted with human cord blood CD34+hematopoietic stem cells (FIGS. 31A and B). In both the marrow as wellthe spleen, the majority of human cells were found to express Venus,while no Venus expression was observed in mouse cells (FIG. 31C, FIG. 37and FIG. 38). Since the mouse genome does not contain an AAVS1 locuscomplementary to the homology arms, these findings demonstrate thespecificity of gene targeting. Of the human cells analyzed, Venusexpression was observed in primitive CD34+ progenitor cells, as well asmature glycophorin A+ erythroid cells in both the marrow as well thespleen.

In another experiment, mice were engrafted with human cord blood CD34+cells and AAVF-Venus was injected by an intravenous route either 1 or 7weeks later. Vertebral or femoral marrow or spleen was harvested either5, 6 or 13 weeks after intravenous injection of Venus. These representedcumulative times post-transplant of 6, 7 or 20 weeks. Results revealthat intravenous injection of AAVF-Venus results in editing of both theprimitive (CD34+) as wells as the more mature, differentiated (CD45+) invivo engrafted human hematopoietic cells. Cells of the human erythroidlineage demonstrated very efficient editing long-term aftertransplantation and injection (FIG. 32 ). AAVF-mediated editing wasfound to be stable long term, and was stably inherited by thedifferentiated progeny of in vivo engrafted human CD34+ cells (FIG. 32). The differentiated progeny of edited CD34+ cells expressed Venus longterm (FIG. 32 ).

In another experiment, sequence analysis was performed for targetedchromosomal insertion of a promoterless SA/2A venus ORF in a K562erythroleukemia cell line, primary human cytokine-primed peripheralblood CD34+ cells and a HepG2 human liver cell line (FIG. 33 ).Site-specifically integrated sequences were amplified using achromosome-specific primer and an insert-specific primer. Resultsrevealed precise insertion of the SA/2A Venus at the junction betweenthe left and right homology arms in every case (FIG. 33 ).

In another experiment, sequence analysis was performed for targetedchromosomal insertion of a 10 bp insert in primary human cytokine-primedperipheral blood CD34+ cells and a HepG2 human liver cell line (FIG. 34). Site-specifically integrated sequences were amplified using achromosome-specific primer and an insert-specific primer. Resultsrevealed precise insertion of the 10 bp insert at the junction betweenthe left and right homology arms in every case (FIG. 34 ).

These data show that both large and short inserts can be successfullyedited into a genome using AAV clade F vectors and that the integrationinto the genome is precise. These data also show that AAV clade Fvectors could be used for high efficiency genome editing in the absenceof an exogenous nuclease.

Example 5: Editing of the PPP1R12c Locus in Human Cell Lines

Methods

To assess the editing of human cell types by AAVF vectors, the followinghuman cell lines were used:

Cell Line Tissue type WI-38 normal human diploid fibroblasts MCF7 humanbreast cancer cell line Hep-G2 human hepatocellular carcinoma cell lineK562 CD34+ erythroleukemic cell line Y79 human retinoblastoma cell lineSCID-X1 human EBV-immortalized B cell line LBL from a SCID-X1 patient

The AAVF vectors used each contained a vector genome containing anediting element encoding a promoterless Venus reporter. The promoterlessVenus contained the open reading frame (ORF) of Venus downstream from asplice acceptor (SA) and a 2A sequence (2A) to allow for independentprotein expression. The left and right homology arms (HA) were each 800bp long and were complementary to sequences in Intron 1 of humanPPP1R12C located in the AAVS1 locus on chromosome 19. The AAVS1 locus isconsidered a safe harbor site for the insertion of heterologoustransgenes. The editing element containing the homology arms, the VenusORF, and regulatory sequences were cloned between AAV2 inverted terminalrepeats (ITRs).

Cell Culture. All cell lines were grown in a humidified atmosphere of 5%CO2 at 37° C. and cultured as follows: SCID-X1 lymphoblasts (Coriell)were cultured in RPMI1640 (Gibco, cat #21875) supplemented with 15%fetal bovine serum (FBS) (Gibco, cat #26140); K562 cells (ATCC) werecultured in DMEM (Corning, cat #15-017-CVR) supplemented with 10% FBS(Gibco, cat #26140) and 1% L-glutamine (Gibco, cat #25030); HepG2 cells(ATCC) were cultured in EMEM (ATCC, cat #30-2003) supplemented with 10%FBS (Gibco, cat #26140); MCF-7 cells (ATCC) were cultured in MEM (Gibco,cat #11095) supplemented with 10% FBS (Gibco, cat #26140), 1% MEMnon-essential amino acids (Gibco, cat #11140), 1% Sodium pyruvate(Gibco, cat #11360) and 10 μg/ml human recombinant insulin (Gibco, cat#12585-014); WI-38 fibroblasts (ATCC) and HEK293 cells (ATCC) werecultured in MEM (Gibco, cat #11095) supplemented with 10% FBS (Gibco,cat #26140), 1% MEM non-essential amino acids (Gibco, cat #11140) and 1%Sodium pyruvate (Gibco, cat #11360); and Y79 cells (ATCC) were culturedin RPMI1640 (Gibco, cat #A10491) supplemented with 20% FBS (Gibco, cat#26140).

AAVF-mediated editing of human cell lines. Adherent cells were seeded onday 0 at 20,000 cells/0.1 mL for a 96-well format or 20,000 cells/0.5 mLfor a 24-well format. Cell counts were measured 24-h later (on day 1)prior to addition of the AAVF vectors. Suspension cells were seeded onday 1 on 20,000 cells/0.1 mL in a 96-well format or 20,000 cells/0.5 mLin 24-well plate format. On day 1, the AAVFs were added to the cellsusing a multiplicity of infection (MOI) of 150,000 vector genomes(VG)/cell. Before AAVF addition, the pipet tips used for transfer werecoated with protamine sulfate (10 mg/ml). The AAVFs were thoroughlysuspended on a vortex mixer at full speed for 30 seconds immediatelybefore transduction. The volume of AAVF added to the cells did notexceed 5% of the total volume in the well. On day 3, cells wereharvested (adherent cells were mildly trypsinized to remove them fromthe tissue culture plates), washed, and analyzed for Venus expression byflow cytometry using an Intellicyt flow cytometer fitted with a HypercytAutosampler. Editing was expressed as the percent of the total cellpopulation that was Venus positive minus the background fluorescenceobserved in untransduced cells (typically less than 1% Venus positivecells). The AAVF editing experiments were carried out without the use ofan exogenous nuclease.

Results

Editing of the human PPP1R12c locus was observed for all the AAVF testedand showed cell type selectivity (Table 3). In general, AAVF5 producedthe highest levels of editing in each of the cell lines, from 12-45% ofthe total cell population, after 48 hours of infection. AAVF9 alsoproduced high levels of gene editing in the B lymphoblast cell lines(SCID-X1 LBL and K562). AAVF17 produced the highest level of geneediting in normal human diploid fibroblasts (WI-38 cells) under theseconditions. The AAVF1, AAVF4, AAVF7 vectors produced levels of editingthat were consistently greater than that seen in untransduced cells butthat were generally lower than the maximal levels observed with AAVF5,AAVF9, and AAVF17. These data demonstrate that the AAVFs have a broadtropism for human tissues and may be useful for gene editing in theliver, CNS (e.g., retina), tissue fibroblasts, breast, and lymphocytesamong others.

TABLE 3 Editing of Human Cell Lines by AAVF Human Cell Lines K562 Hep-G2WI-38 Y79 MCF7 SCID-X1 LBL Vector Percent Venus Positive Cells AAVF12.06 1.92 1.39 0.7 2.71 0.57 AAVF4 1.36 2.95 6.29 0.35 3.42 0.12 AAVF545.32 — 11.85 12.5 20.47 45.02 AAVF7 5.78 3.83 4.87 1.42 3.6 0.94 AAVF918.06 — — 2.46 6.14 6.79 AAVF17 3.18 3.71 17.6 1.29 5.66 0.44

Example 6: AAVF Editing in Primary Human Cells

To assess the editing of primary human cells by AAVF vectors, primarycultures of human hepatocytes, hepatic sinusoidal endothelial cells, andskeletal muscle myoblasts were used.

The AAVF and AAV vectors used each packaged a vector genome encoding apromoter-less Venus reporter. The insert comprised a Venus open readingframe (ORF) downstream from a splice acceptor (SA) and a 2A sequence(2A) to allow for independent protein expression. The left and righthomology arms (HA) were each 800 bp long and were complementary tosequences in Intron 1 of human PPP1R12C located in the AAVS1 locus onchromosome 19. The AAVS1 locus is considered a safe harbor site for theinsertion of heterologous transgenes. The correction genome consistingof the homology arms, the open reading frame and regulatory sequenceswere cloned between AAV2 inverted terminal repeats (ITRs).

Methods

Cell culture. All primary human cells were cultured at 37° C., under 5%humidified CO2 in a tissue culture incubator. All materials and mediacomponents were obtained from Life Technologies unless specifiedotherwise.

Primary cultures of human hepatocytes were obtained from Invitrogen andwere cultured on type I collagen coated plates as suggested by themanufacturer. Cells were recovered from storage in liquid nitrogen inThawing/Plating Medium [32.5 mL William's E Medium (#A12176) 1.6 mLfetal bovine serum, 3.2 uL of 10 mM Dexamethazone and 0.9 mL of PlatingCocktail A per 35.0 mL final volume]. Plating cocktail A consisted of0.5 mL Penicillin (10,000 U/mL)/Streptomycin (10,000 ug/mL) solution(Cat #15140), 0.05 mL of 4.0 mg human recombinant insulin/mL (Catalog#12585-014), 0.5 mL of 200 mM GlutaMAX™ solution (Catalog #35050) and0.75 mL of 1.0M Hepes, pH7.4 (Catalog #15630) per 1.8 mL final volume.Human hepatocytes were maintained in Maintenance media which contained100 mL of Williams E Medium, 0.001 mL of dexamethasone, and 3.6 mL ofMaintenance Cocktail B (Catalog #A13448) per 103.6 mL final volume.

Primary cultures of human skeletal muscle myoblasts were obtained fromLonza and were cultured in SkGM™ medium as described by themanufacturer.

SkGM™-2 Bullit™ Kit (Lonza, Catalog No. CC-3245) contained 0.5 mL humanEpidermal Growth Factor [hEGF] (#0000482653), 0.5 mL Dexamethasone(#0000474738), 10 mL L-glutamine (#0000474740), 50 mL Fetal Bovine Serum(#0000474741), 0.5 mL Gentamicin/Amphotericin-B [GA] (#0000474736), in500 mL SkGM-2 medium (#0000482653).

Primary cultures of human hepatic sinusoidal endothelial cells werepurchased from Creative Bioarray and were cultured in SuperCult®Endothelial Cell Medium and grown on a gelatin-based coating asdescribed by the manufacturer. SuperCult® Endothelial Cell MediumSupplement Kit (Catalog #ECM-500 Kit) contained 0.5 mL VEGF (#15206),0.5 mL Heparin (#15250), 0.5 mL EGF (#15217), 0.5 mL FGF (#15204), 0.5mL Hydrocortisone (#15318), 5.0 mL Antibiotic-Antimycotic Solution(#15179), 5.0 mL L-glutamine (#15409), 10.0 mL Endothelial CellSupplement (#15604), 50.0 mL FBS (#15310), and 500.0 mL of SuperCult®Endothelial Cell Medium (#15517).

AAVF-mediated editing of primary human cells. Human primary hepatocyteswere seeded on day 0 on 2×10⁴ cells/0.1 mL for the 96-well format or2×10⁴ cells/0.5 mL for the 24-well format. Viral vectors were added 48 hlater on day 2 as described below. Human skeletal muscle myoblasts, andhuman hepatic sinusoidal endothelial cells were seeded on day 1 at 2×10⁴cells/0.1 mL in a 96-well format or 2×10⁴ cells/0.5 mL in a 24-wellformat. On day 2, the viral vectors were added to these cells at amultiplicity of infection (MOI) of 150,000 VG (Vector genomes)/cell (ForAAVF5 an MOI of 5×10⁴ VG/cell was used and for AAVF17 an MOI of 2.5×10⁴VG/cell was used). Prior to the addition of vector to the cells, allpipet tips used for vector transfer were coated with protamine sulfate(10 mg/mL) and the vectors were thoroughly mixed by vortexing for 30seconds immediately before transduction. The volume of vector added tothe cells did not exceed 5% of the total volume in the well.

The culture media for the human primary hepatocytes was refreshed on day3. On day 4, cells were harvested (adherent cells were trypsinized) andanalyzed for Venus expression by flow cytometry using an Intellicyt flowcytometer fitted with a Hypercyt Autosampler. Editing was expressed asthe percent of the total cell population that was Venus positive minusthe background fluorescence observed in un-transduced cells (typicallyless than 1% Venus positive cells).

Results

Editing of the human PPP1R12c locus was observed for all the AAVF testedand showed cell type selectivity (Table 4). In general, AAVF5 gave thehighest levels of editing in each of the primary cell populations, froma low of 2% in human hepatocytes to 24% to 35% in primary skeletalmyoblasts and hepatic sinusoidal endothelial cells, respectively, after48-h of infection. AAVF7 and AAVF17 also produced high levels of geneediting in the primary hepatic sinusoidal endothelial cells and skeletalmyoblasts. Levels of editing with the AAVF vectors in these cells were10- to 50-fold higher than that observed with AAV2 or AAV6. The AAVFvectors produced levels of editing that were consistently greater thanthat seen in untransduced cells or cells transduced with a AAV2 or AAV6packaging the promoter-less Venus vector genome. Little or no editingwas observed for either AAV2 or AAV6 in these cells. These data inprimary human cells demonstrate that AAVF5, AAVF7, and AAVF17 have abroad tropism for human tissues and may be useful for gene therapyapplications directed towards the liver, skeletal muscle, andendothelial cell populations, among others.

For comparison, each of the primary human cells populations were alsotransduced with AAVF gene transfer vectors packaging mCherry undercontrol of the chicken beta actin (CBA) promoter. The ratios of proteinexpression of Venus/mCherry were then used to estimate an editingratio—reflecting the ratio of number of cells having detectable proteinexpression in the various cell types using gene editing vectors of theexperiment (Venus) to number of cells having detectable proteinexpression using the aforementioned gene transfer vectors (mCherry)—forAAV6, AAVF5, and AAVF7. As shown in Table 5, AAVF-mediated gene editingis generally more effective for protein expression than thecorresponding AAVF-mediated gene transfer approach in the studiedprimary human cells, whereas AAV6-mediated gene editing wassubstantially the same as or slightly less effective than AAV6-mediatedgene transfer in such cells. Notably, as also shown in Table 5,AAVF-mediated gene editing was higher than that observed withAAV6-mediated gene editing for the studied primary human cells. Thesedata demonstrate that AAVF-mediated gene editing is more efficient thanAAV6 in a variety of primary human cells, and that AAVF-mediated geneediting compares favorably over corresponding gene-transfer approachesin such cells.

The AAVF vectors packaging the mCherry reporter also effectivelytransduced primary human cells and showed cell type specificity (Table6). For human umbilical vein endothelial cells (HUVEC) and hepaticsinusoidal endothelial cells (HSEC), transduction with AAVF9 produced38-50% mCherry positive cells after 48-h of infection. AAVF9 alsoefficiently transduced human skeletal muscle myoblasts and to a lesserextent, all of the AAVF tested effectively transduced the HSEC underthese conditions (Table 6). Without being bound by theory, it is likelythat most, if not all, of the mCherry expression in these studiesrepresents episomal expression of the reporter as no homology arms werepresent in the mCherry vector genomes with expression was driven by theCBA promoter.

TABLE 4 AAVF-Mediated Editing of Primary Human Cells HSEC HepatocytesSMM Vector Percent Venus Positive Cells AAV2 0.34 0.06 0.52 AAV6 0.000.00 0.00 AAVF5 34.70 2.00 24.20 AAVF7 18.40 0.62 18.65 AAVF17 21.401.37 19.60

TABLE 5 Editing ratios of AAVF in primary human cells Hepatocytes SMMHSEC Venus/ AAVF/ Venus/ AAVF/ Venus/ AAVF/ Vector mCherry AAV6 mCherryAAV6 mCherry AAV6 AAV6 0.84 0.80 0.60 AAVF5 9.42 11.21 14.32 17.90 5.308.80 AAVF7 0.95 1.10 13.05 16.30 2.70 4.50HSEC=hepatic sinusoidal endothelial cellsSMM=skeletal muscle myoblasts

TABLE 6 Tranduction of Primary Human Cells with AAVF Vectors PackagingmCherry Vector HSEC Hepatocytes SMM HUVEC Percent mCherry positive cellsAAVF1 7.04 0.13 1.65 1.47 AAVF4 9.10 0.05 1.72 0.61 AAVF5 4.00 0.01 1.030.71 AAVF7 4.43 0.70 0.77 2.15 AAVF9 38.24 1.05 29.27 50.13 HSEC =hepatic sinusoidal endothelial cells SMM = skeletal muscle myoblastsHUVEC = human umbilical vein endothelial cells

Example 7: Study of AAVF Relative Gene-Editing (HDR) VersusGene-Transfer (Transduction) Efficiencies

Two types of AAV-based vectors (CBA-mCherry gene transfer vectors, andAAVS1-Venus gene editing vectors, FIG. 39 ) were used to assess therelative gene-editing efficiency versus the gene-transfer transductionefficiency of AAVF vectors, as well as AAV2 and AAV6, as measured byrelative protein expression. For the gene-transfer vectors, CBA-mCherryconstruct included the mCherry gene under the control of the chickenbeta actin (CBA) promoter, and utilized a polyadenylation signal, butdid not contain any homology arms. For the gene-editing vectors,AAVS1-Venus construct included the promoterless Venus open reading frame(ORF), with (HA-L) and right (HA-R) homology arms targeting the VenusORF to Intron 1 of the PPP1R12C gene within the AAVS1 region ofchromosome 19. There was also a splice acceptor (SA) and a 2A sequenceupstream of the Venus ORF, which allowed the Venus transcript to bespliced out and expressed independent of the PPP1R12C gene.

The ability of AAVF, AAV2 and AAV6 vectors to mediate gene editingversus gene transfer was compared in primary human cord blood CD34+hematopoietic stem/progenitor cells. The cells used were pooled frommultiple donors. Purified CD34+ cells were transduced with either genetransfer vectors (AAV*-CBA-mCherry) or gene editing vectors (AAV*-Venus)at a multiplicity of 150,000 vector genomes (VG) per cell. Forty eighthours later, cells were harvested and analyzed by flow cytometry (FIGS.40A and 40B). The data shown includes subtraction of backgrounduntransduced cells.

It was hypothesized that mCherry expression would represent transductionefficiency, whereas Venus expression would represent gene editingefficiency. Without being bound by theory, the following is a summary ofthe potential mechanism of AAV transduction versus editing. Uponinfection, AAV binds to cell surface receptors and is internalized priorto nuclear translocation and entry. In the nucleus, the AAV undergoesuncoating and vector genomes are released. These processes likely occurat the same rate for each given capsid in the same cell population,regardless of the vector genome. Following uncoating, the singlestranded CBA-mCherry genome undergoes second strand synthesis prior tomCherry expression. On the other hand, the promoterless Venus editingvector is directed to the genomic region of complementarity onChromosome 19. The SA/2A-Venus cassette which is bounded by the homologyarms may then recombine into the chromosome at the internal junction ofthe homology arms via homology dependent repair (HDR) mechanisms.Following this recombination event, Venus is expressed in thesuccessfully edited cells.

Venus was expressed in a much higher proportion of cells than mCherryfor all vectors except AAVF9 (FIGS. 40A and 40C). Venus expression wasespecially high following transduction with AAVF1, AAVF5, AAVF7, AAVF16and AAVF17. The same capsids led to much lower levels of mCherryexpression (FIGS. 40A and 40C). The least amount of both mCherry andVenus expression was observed following transduction with AAV2 and AAV6.A comparison of the relative expression of Venus to mCherry was alsoperformed. Specifically, an editing ratio—reflecting the ratio of numberof cells having detectable protein expression in the various cell typesusing gene editing vectors of the experiment (Venus) to the number ofcells having detectable protein expression using the aforementioned genetransfer vectors (mCherry)—was determined. The editing ratio provides anestimate of the relative efficiency of editing mediated by each AAVcapsid after normalization for the processes of virus entry throughuncoating within the nucleus. All AAV vectors tested exhibited moreefficient gene editing (Venus) as compared with gene transfer/transgeneexpression (mCherry), except for AAVF9 (FIG. 40D and Table 7). AAVF5 andAAVF7 displayed the highest editing ratio (FIG. 40D and Table 7).AAVF-mediated gene editing (Venus) was also compared relative to AAV 2-and AAV6-mediated gene editing (Venus) (Table 7, which shows theVenus:mCherry ratio of AAVF divided by the same ratio for either AAV2 orAAV6). The gene-editing effectiveness of AAVF gene-editing constructscompared favorably relative to AAV2- and AAV6-gene editing constructs.The editing ratios of AAVF5 and AAVF7 were the highest of the vectorscompared, indicating that these vectors in particular mediate highlyefficient editing.

TABLE 7 Editing-to-Transduction ratio in CD34+ CB Cells Ratio editing:Ratio Ratio transduction AAVF editing: AAVF editing: (Venus: AAV6 AAV2Vector mCherry) editing editing AAVF1 4.32 1.23 3.02 AAVF4 3.42 0.980.98 AAVF5 16.60 4.74 11.62 AAVF7 14.66 4.19 10.26 AAVF9 0.73 0.21 0.51AAV2 1.43 0.41 1.00 AAV6 3.50 1.00 2.45

The present invention is not to be limited in scope by the specificembodiments disclosed in the examples which are intended asillustrations of a few aspects of the invention and any embodiments thatare functionally equivalent are within the scope of this invention.Indeed, various modifications of the invention in addition to thoseshown and described herein will become apparent to those skilled in theart and are intended to fall within the scope of the appended claims.

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1-146. (canceled)
 147. A replication-defective adeno-associated virus(AAV) comprising: (a) a correction genome comprising (i) an editingelement for integration into a target locus of a chromosome in a cell,the editing element comprising a coding sequence of a gene optionallyoperably linked to an exogenous promoter, (ii) a 5′ homologous armnucleotide sequence 5′ of the editing element, having homology to a 5′region of the chromosome relative to the target locus, and (iii) a 3′homologous arm nucleotide sequence 3′ of the editing element, havinghomology to a 3′ region of the chromosome relative to the target locus;and (b) an AAV capsid comprising an AAV capsid protein comprising theamino acid sequence of amino acids 203-736 of SEQ ID NO:
 13. 148. Thereplication-defective AAV of claim 147, wherein the editing elementcomprises a polyadenylation sequence operably linked to the codingsequence.
 149. The replication-defective AAV of claim 147, wherein eachof the 5′ and 3′ homologous arm nucleotide sequences independently has anucleotide length of about 50 to 2000 nucleotides.
 150. Thereplication-defective AAV of claim 147, wherein the target locus is anintron.
 151. The replication-defective AAV of claim 147, wherein thecorrection genome comprises a 5′ inverted terminal repeat (5′ ITR)nucleotide sequence 5′ of the 5′ homologous arm nucleotide sequence, anda 3′ inverted terminal repeat (3′ ITR) nucleotide sequence 3′ of the 3′homologous arm nucleotide sequence.
 152. The replication-defective AAVof claim 151, wherein the 5′ ITR nucleotide sequence has at least 95%sequence identity to SEQ ID NO: 36 or 38, and the 3′ ITR nucleotidesequence has at least 95% sequence identity to SEQ ID NO: 37 or
 39. 153.The replication-defective AAV of claim 147, wherein the correctiongenome is a single stranded genome.
 154. A replication-defective AAVcomprising: (a) a correction genome comprising (i) an editing elementfor integration into a target locus of a chromosome in a cell, theediting element comprising a coding sequence of a gene optionallyoperably linked to an exogenous promoter, (ii) a 5′ homologous armnucleotide sequence 5′ of the editing element, having homology to a 5′region of the chromosome relative to the target locus, and (iii) a 3′homologous arm nucleotide sequence 3′ of the editing element, havinghomology to a 3′ region of the chromosome relative to the target locus;and (b) an AAV capsid comprising an AAV capsid protein comprising theamino acid sequence of amino acids 138-736 of SEQ ID NO:
 13. 155. Thereplication-defective AAV of claim 154, wherein the editing elementcomprises a polyadenylation sequence operably linked to the codingsequence.
 156. The replication-defective AAV of claim 154, wherein eachof the 5′ and 3′ homologous arm nucleotide sequences independently has anucleotide length of about 50 to 2000 nucleotides.
 157. Thereplication-defective AAV of claim 154, wherein the target locus is anintron.
 158. The replication-defective AAV of claim 154, wherein thecorrection genome comprises a 5′ ITR nucleotide sequence 5′ of the 5′homologous arm nucleotide sequence, and a 3′ ITR nucleotide sequence 3′of the 3′ homologous arm nucleotide sequence.
 159. Thereplication-defective AAV of claim 158, wherein the 5′ ITR nucleotidesequence has at least 95% sequence identity to SEQ ID NO: 36 or 38, andthe 3′ ITR nucleotide sequence has at least 95% sequence identity to SEQID NO: 37 or
 39. 160. The replication-defective AAV of claim 154,wherein the correction genome is a single stranded genome.
 161. Areplication-defective AAV comprising: (a) a correction genome comprising(i) an editing element for integration into a target locus of achromosome in a cell, the editing element comprising a coding sequenceof a gene optionally operably linked to an exogenous promoter, (ii) a 5′homologous arm nucleotide sequence 5′ of the editing element, havinghomology to a 5′ region of the chromosome relative to the target locus,and (iii) a 3′ homologous arm nucleotide sequence 3′ of the editingelement, having homology to a 3′ region of the chromosome relative tothe target locus; and (b) an AAV capsid comprising an AAV capsid proteincomprising the amino acid sequence of SEQ ID NO:
 13. 162. Thereplication-defective AAV of claim 161, wherein the editing elementcomprises a polyadenylation sequence operably linked to the codingsequence.
 163. The replication-defective AAV of claim 161, wherein eachof the 5′ and 3′ homologous arm nucleotide sequences independently has anucleotide length of about 50 to 2000 nucleotides.
 164. Thereplication-defective AAV of claim 161, wherein the target locus is anintron.
 165. The replication-defective AAV of claim 161, wherein thecorrection genome comprises a 5′ ITR nucleotide sequence 5′ of the 5′homologous arm nucleotide sequence, and a 3′ ITR nucleotide sequence 3′of the 3′ homologous arm nucleotide sequence.
 166. Thereplication-defective AAV of claim 165, wherein the 5′ ITR nucleotidesequence has at least 95% sequence identity to SEQ ID NO: 36 or 38, andthe 3′ ITR nucleotide sequence has at least 95% sequence identity to SEQID NO: 37 or
 39. 167. The replication-defective AAV of claim 161,wherein the correction genome is a single stranded genome.
 168. Apackaging system for recombinant preparation of a replication-defectiveAAV, wherein the packaging system comprises (a) a Rep nucleotidesequence encoding one or more AAV Rep proteins; (b) a Cap nucleotidesequence encoding an AAV capsid protein as set forth in claim 147; and(c) a correction genome as set forth in claim 147, wherein the packagingsystem is operative in a cell for enclosing the correction genome in thecapsid to form the replication-defective AAV.
 169. A method forrecombinant preparation of a replication-defective AAV, the methodcomprising introducing the packaging system of claim 168 into a cellunder conditions operative for enclosing the correction genome in thecapsid to form the replication-defective AAV.
 170. A method for editinga target locus of a genome in a cell, the method comprising transducingthe cell with the replication-defective AAV of claim 147.